Compositions and methods to treat cancer with CpG rich DNA and cupredoxins

ABSTRACT

The present invention relates to compositions comprising CpG rich DNA from  Pseudomonas aeruginosa . The compositions optionally comprise a cupredoxin. The present invention includes specific CpG DNAs from  Pseudomonas aeruginosa  that are useful for treating cancer and other conditions in patients. These compositions are optionally in a pharmaceutically acceptable carrier and also optionally comprise a cupredoxin. The present invention further relates to methods to express proteins near cancer cells. These methods may be used to express therapeutic or diagnostic proteins near cancer cells in a patient suffering from cancer or other conditions, and can also be used for diagnosing cancer in a patient. This method uses the gene for azurin from  P. aeruginosa  as an expression system for azurin or heterologous proteins in  P. aeruginosa  or heterologous cells.

RELATED APPLICATIONS

This application is a divisional and claims the benefit, under 35 U.S.C.§ 121, of U.S. patent application Ser. No. 11/950,165, filed Dec. 4,2007, which issued as U.S. Pat. No. 8,017,749, and which claims priorityunder 35 U.S.C. §§ 119 and 120 to U.S. Provisional Patent ApplicationSer. No. 60/872,471, filed Dec. 4, 2006.

STATEMENT OF GOVERNMENTAL INTEREST

This invention was made with government support under a research grantfrom the National Institutes of Health (NIH), Bethesda, Md., U.S.A.,(Grant Numbers AI 16790-21, ES 04050-16, AI 45541, CA09432 andN01-CM97567). The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to compositions comprising CpG rich DNAfrom Pseudomonas aeruginosa and, optionally, cupredoxins. The presentinvention includes specific CpG DNAs from Pseudomonas aeruginosa thatare useful for treating patients, particularly suffering fromconditions, specifically cancer, and preventing cancer. Thesecompositions are optionally in a pharmaceutically acceptable carrier andoptionally comprise a cupredoxin. The present invention further relatesto methods to express proteins near cancer cells. These methods may beused to express therapeutic or diagnostic proteins near cancer cells ina patient suffering from conditions, and specifically cancer, preventingcancer, and diagnosing cancer in a patient. This method uses the genefor azurin from P. aeruginosa as an expression system for azurin orheterologous proteins in P. aeruginosa or heterologous cells.

BACKGROUND OF THE INVENTION

While the use of live bacteria in DNA vaccination and gene therapy hasshown significant potential in cancer therapy, another avenue has beenthe use of bacterial products such as lipolysaccharides, peptidoglycansor even naked DNA. Vassaux et al., J. Pathol. 208:290-298 (2006). Asearly as in 1984, the DNA from Mycobacterium bovis BCG was shown to haveantitumor properties. Indeed, Tokunaga et al. described the isolation ofpurified DNA from BCG that demonstrated significant tumor regression inmice and substantial regressing response in human skin malignancies.Tokunaga et al., Japan J. Infect. Dis. 52:1-11 (1999).

The immunostimulatory activity leading to tumor regression in the BCGDNA is due to the presence of DNA stretches rich in unmethyalted CpGdinucleotides. Unmethylated CpG sequences are 20 times more common inbacterial DNA as compared to mammalian DNA, and the presence of specificunmethylated CpG sequences (motifs) are recognized by the Toll-likereceptor (TLR) 9. This interaction between the bacterial CpG DNA motifand TLR9 activates the monocytes and dendritic cells to produceinterleukin-12 which in turn activates the T-helper 1 cells. In contrastto CpG DNA, bacterial lipopolysaccharides are recognized by TLR4 whilethe corresponding lipoproteins are recognized by TLR2. Modlin, Nature408:659-660 (2000); Krieg, Nature Med. 9:831-835 (2003). Since thebacterial DNA rich in unmethylated CpG sequences is difficult to purifyfor human clinical trials, synthetic oligodeoxynucleotides (ODNs) of 8to 30 bases and containing one or more CpG motifs, have been used withencouraging results in the immunotherapy of viral, bacterial andparasitic infections, as well as in limited phase I human clinicaltrials in patients with basal cell carcinoma or melanoma. Krieg, Id.

In contrast to bacterial CpG motifs, mammalian cell DNA contains CpGsequences where the cytosine residues are highly methylated. Forexample, about 6 to 8% of human DNA cytosine residues are believed to bemethylated by DNA methyltransferases whose levels are modestly higher inhuman tumors than in normal cells. The promoter regions of a number ofhuman genes have CpG islands that are hypermethylated leading tosilencing of the downstream genes. Such epigenetic silencing of thedownstream genes, particularly in tumor suppressor genes such asp16Ink4a, BRCA1 or hMLH1, triggers tumor formation and where developmentof specific DNA-demethylating agents can serve as antitumor drugs.Herman and Baylin, New Eng. J. Med. 349:2042-2054 (2003); Feinberg andTycko, Nature Rev. Cancer 4:143-153 (2004). Transcriptional profiling ofseveral breast cancer cell lines has demonstrated the presence of CpGislands that are highly methylated in the primary tumors and in the celllines but not in the DNA from normal breast epithelia or matchedlymphocytes from cancer patients. Treatment of a breast cancer cell linewith the DNA methylation inhibitor 5-azacytidine resulted in the growtharrest of the cancer cells, demonstrating the importance of promoter CpGisland methylation in tumor growth. Wang et al., Oncogene 24:2705-2714(2005).

What is needed are new treatments for patients, specifically patientssuffering from conditions, and specifically cancer, and preventativetreatments for cancer. Such treatments may be new treatment methods, orvariations or improvements on previously described treatment methods.Such cancer treatments should be able to slow the growth of tumors inmammalian patients, and/or decrease the size of tumors in mammalianpatients. Also needed are methods to deliver therapeutic molecules tocancer cells in a patient as well as diagnose the existence and locationof tumors in patients.

SUMMARY OF THE INVENTION

The present invention relates to compositions comprising CpG rich DNApolynucleotides from Pseudomonas aeruginosa. Specifically, thesecompositions may comprise CpG rich DNA from P. aeruginosa, andoptionally a cupredoxin peptide, such as azurin from Pseudomonasaeruginosa, and/or the 50-77 residue region of azurin (p28). Theinvention further relates to the use of the compositions of theinvention to treat patients, particularly mammalian patients sufferingfrom conditions, and specifically cancer, and to prevent cancer. Thepresent invention further relates to cells and methods to express aprotein upon contact with cancer cells, and methods to administer thecells to diagnose and treat cancer.

In one aspect of the invention, an isolated nucleic acid moleculecomprising a polynucleotide sequence selected from the group consistingof (a) a sequence selected from the group consisting of SEQ ID NOS:26-62 and (b) a sequence that is at least 90% identical to a sequenceselected from the group consisting of SEQ ID NOS: 26-62. In a specificembodiment, the polynucleotide is SEQ ID NO: 26. The isolated nucleicacid sequence may be in a pharmaceutically acceptable carrier. Thispharmaceutical composition may also comprise at least one cupredoxinpeptide. In some embodiments, the pharmaceutical composition isformulated for intravenous, subcutaneous or topical administration. Insome embodiments, the cupredoxin peptide in the pharmaceuticalcomposition may be from an organism selected from the group consistingof Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacterxylosoxidan Bordetella bronchiseptica, Methylomonas sp., Neisseriameningitidis, Neisseria gonorrhea, Pseudomonas fluorescens, Pseudomonaschlororaphis, Xylella fastidiosa, and Vibrio parahaemolyticus. In someembodiments, the cupredoxin peptide in the pharmaceutical compositionmay be part or all of a protein selected from the group consisting of anazurin, pseudoazurin, plastocyanin, rusticyanin, Laz, auracyanin,stellacyanin or cucumber basic protein. The pharmaceutical compositionmay also comprise part of all of a peptide selected from the groupconsisting of SEQ ID NOS: 1-25.

The present invention also includes methods. Any of the methodsaccording to the present invention can be used to treat a patientsuffering from a condition. In certain embodiments of the methodsaccording to the present invention, the patient is suffering from acancer.

Cancers treated in accordance with the present invention can include,without limitation melanoma, breast, pancreas, glioblastoma,astrocytoma, lung, colorectal, neck and head, bladder, prostate, skin,and cervical cancer.

Administration routes can include, without limitation, intravenousinjection, intramuscular injection, subcutaneous injection, inhalation,topical administration, transdermal patch, suppository, vitreousinjection and oral.

In one embodiment the method includes a method to treat a patient,comprising administering the patient an isolated nucleic acid moleculecomprising a polynucleotide sequence selected from the group consistingof (a) a sequence selected from the group consisting of SEQ ID NOS:26-62 and (b) a sequence that is at least 90% identical to a sequenceselected from the group consisting of SEQ ID NOS: 26-62 wherein theisolated nucleic acid molecule is in a pharmaceutically acceptablecarrier and co-administering a cupredoxin peptide.

In another embodiment of the methods according to the present invention,the method comprises administering to the patient a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier, at leastone cupredoxin peptide and an isolated nucleic acid molecule comprisinga polynucleotide sequence selected from the group consisting of (a) asequence selected from the group consisting of SEQ ID NOS: 26-62 and (b)a sequence that is at least 90% identical to a sequence selected fromthe group consisting of SEQ ID NOS: 26-62.

In another embodiment of the methods according to the present invention,the pharmaceutical composition is administered by a mode selected fromthe group consisting of intravenous injection, intramuscular injection,subcutaneous injection, inhalation, topical administration, transdermalpatch, suppository, vitreous injection and oral.

In another embodiment of the methods according to the present invention,the method comprises administering the patient an isolated nucleic acidmolecule comprising a polynucleotide sequence selected from the groupconsisting of (a) a sequence selected from the group consisting of SEQID NOS: 26-62 and (b) a sequence that is at least 90% identical to asequence selected from the group consisting of SEQ ID NOS: 26-62 whereinthe isolated nucleic acid molecule is in a pharmaceutically acceptablecarrier and co-administering a cupredoxin peptide and furtherco-administering an additional prophylactic or therapeutic drug.

The present invention also includes cells. In one embodiment the cellharbors a heterologous gene for azurin from Pseudomonas aeruginosa,wherein the cell expresses the azurin upon contact with cancer cells. Inanother embodiment, the coding sequence for azurin in the heterologousgene for azurin has been replaced with a coding sequence for a targetprotein, wherein the cell expresses the target protein upon contact withcancer cells. In another embodiment the cell is a Pseudomonas aeruginosacell, wherein the coding sequence for azurin in the genome has beenreplaced with a coding sequence for a target protein, and whichexpresses the target protein upon contact with cancer cells. In anotherembodiment, wherein the coding sequence for azurin in the heterologousgene for azurin has been replaced with a coding sequence for a targetprotein and wherein the cell expresses the target protein upon contractwith cancer cells, the target protein is selected from the groupconsisting of a prophylactic protein, a therapeutic protein, a cytotoxicprotein, and a diagnostic protein.

The present invention also includes methods of using the cells describedin the preceding paragraph. In certain of these embodiments, the methodscomprise treating a patient by administering one or more of thedescribed cell types. The present invention also includes methods ofdiagnosing cancer in a patient using the described cell types describedin the preceding paragraph. In one embodiment the method includesadministering a cell that generally harbors a heterologous gene forazurin from Pseudomonas aeruginosa, wherein the cell expresses theazurin upon contact with cancer cells and wherein the coding sequencefor azurin in the heterologous gene for azurin has been replaced with acoding sequence for a target protein, wherein the cell expresses thetarget protein upon contract with cancer cells and wherein the targetprotein is a diagnostic protein. In other embodiments, the targetprotein is selected from the group consisting of a prophylactic protein,a therapeutic protein, a cytotoxic protein, and a diagnostic protein. Inadditional embodiments the pharmaceutical composition can beadministered by a mode selected from the group consisting of intravenousinjection, intramuscular injection, subcutaneous injection, inhalation,topical administration, transdermal patch, suppository, vitreousinjection and oral. Again, with these methods, the patient can besuffering from a condition and/or cancer. These and other aspects,advantages, and features of the invention will become apparent from thefollowing figures and detailed description of the specific embodiments.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1. Amino acid sequence of azurin from Pseudomonas aeruginosa.

SEQ ID NO: 2. Amino acid sequence of p28, Pseudomonas aeruginosa azurinresidues 50-77.

SEQ ID NO: 3. Amino acid sequence of plastocyanin from Phormidiumlaminosum.

SEQ ID NO: 4. Amino acid sequence of rusticyanin from Thiobacillusferrooxidans.

SEQ ID NO: 5. Amino acid sequence of pseudoazurin from Achromobactercycloclastes.

SEQ ID NO: 6. Amino acid sequence of azurin from Alcaligenes faecalis.

SEQ ID NO: 7. Amino acid sequence of azurin from Achromobacterxylosoxidans ssp. denitrificans I.

SEQ ID NO: 8. Amino acid sequence of azurin from Bordetellabronchiseptica.

SEQ ID NO: 9. Amino acid sequence of azurin from Methylomonas sp. J.

SEQ ID NO: 10. Amino acid sequence of azurin from Neisseria meningitidisZ2491.

SEQ ID NO: 11. Amino acid sequence of azurin from Pseudomonasfluorescen.

SEQ ID NO: 12. Amino acid sequence of azurin from Pseudomonaschlororaphis.

SEQ ID NO: 13. Amino acid sequence of azurin from Xylella fastidiosa9a5c.

SEQ ID NO: 14. Amino acid sequence of stellacyanin from Cucumis sativus.

SEQ ID NO: 15. Amino acid sequence of auracyanin A from Chloroflexusaurantiacus.

SEQ ID NO: 16. Amino acid sequence of auracyanin B from Chloroflexusaurantiacus.

SEQ ID NO: 17. Amino acid sequence of cucumber basic protein fromCucumis sativus.

SEQ ID NO: 18. Amino acid sequence of Laz from Neisseria gonorrhoeaeF62.

SEQ ID NO: 19. Amino acid sequence of the azurin from Vibrioparahaemolyticus.

SEQ ID NO: 20. Amino acid sequence of amino acids 57 to 89 of auracyaninB of Chloroflexus aurantiacus.

SEQ ID NO: 21. Amino acid sequence of amino acids 51-77 of Bordetellapertussis azurin.

SEQ ID NO: 22. Amino acid sequence of amino acids 89-115 of Neisseriameningitidis Laz.

SEQ ID NO: 23. Amino acid sequence of amino acids 51-77 of Pseudomonassyringae azurin.

SEQ ID NO: 24. Amino acid sequence of amino acids 52-78 of Vibrioparahaemolyticus azurin.

SEQ ID NO: 25. Amino acid sequence of amino acids 51-77 of Bordetellabronchiseptica azurin.

SEQ ID NOS: 26. DNA sequence of polynucleotide in 15 kb band that isreleased from P. aerugniosa that encodes a polypeptide with similarityto Laz from Neisseria.

SEQ ID NOS: 27-62. DNA sequences of polynucleotides in 15 kb band thatis released from P. aerugniosa.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the secretion of azurin by P. aeruginosa strains 8821 and8822 in the presence of cancer cells. FIG. 1A. Two P. aeruginosa strains(8822, 8821) were grown in the presence (MCF-7) or absence (control) ofcancer cells, and the secretion of azurin in the extracellular fractionwas examined by Western blotting using anti-azurin antibody. Panel a:growth of P. aeruginosa cells by measurement of turbidity at 600 nm.Squares and diamonds indicate the secretion level of azurin in thepresence and absence of cancer cells respectively. The difference in theturbidity at 0 min between the presence and absence of cancer cells isdue to the addition of cancer cells. Panel b: diagrammaticrepresentation of the secretion level of azurin by measurement of thesignal intensity on Western blotting profiles. Squares and diamondsindicate the secretion level of azurin in the presence and absence ofcancer cells respectively. Panel c: western blotting. Extracellularfractions were prepared at 0, 15, 30 and 60 min after the addition ofcancer cells and then subjected to SDS-PAGE, followed by Westernblotting. Arrows indicate the position of azurin. FIG. 1B. QuantitativeWestern blotting. The signal intensity on the Western blotting profileincreases linearly with the increase of standard azurin (0 to 20 ng).FIG. 1C. Production of azurin in P. aeruginosa strains 8821 and 8822.Intracellular fractions from such cells (8821, 8822) harvested atexponential growth phase were subjected to SDS-PAGE, followed by Westernblotting using anti-auzrin antibody. Standard azurin is shown in theleft lane. FIG. 1D. Lack of detection of azurin in MCF-7 cell extracts(100 μg protein) by Western blotting.

FIG. 2 depicts the lack of P. aeruginosa cell lysis in the presence ofcancer cells. FIG. 2A. Strain 8822 cells transformed with plasmid pQF47containing the lacZ gene were grown in the presence (MCF-7) or absence(control) of cancer cells, and extracellular and intracellular fractionswere subjected to SDS-PAGE, followed by Western blotting using anti-LacZantibody. Sample preparation was carried out as in FIG. 1. Standard LacZis shown in the left lane. Asterisk indicates the intracellular fractionof the parental strain 8822 cells (without the pQF47 plasmid) incubatedfor 60 min with cancer cells. FIG. 2B. Extracellular fractions of strain8822 cells transformed with pQF47 were subjected to SDS-PAGE, followedby Western blotting using anti-azurin antibody. FIG. 2C. Functionalexpression of LacZ in strain 8822 cells transformed with pQF47.

FIG. 3 depicts azurin, but not cytochrome c₅₅₁, is secreted from theperiplasmic space of E. coli cells in response to the presence of humanbreast cancer MCF-7 cells in an energy-independent manner. Samplepreparation was carried out as in FIG. 1. Asterisks (0*) indicate theintracellular fraction just before incubation with cancer cells.Positions of azurin and cytochrome c₅₅₁ (cyt c₅₅₁) are indicated byarrows. FIG. 3A, azurin secretion from E. coli JM109; FIG. 3B,cytochrome c₅₅₁ secretion form E. coli JCB7120; FIG. 3C, azurinsecretion from P. aeruginosa 8822 when such cells were preincubated for1 h with 0, 50 or 250 μM CCCP. FIG. 3D, azurin secretion from E. coliJM109 when such cells were preincubated with CCCP at 250 μMconcentrations for 1 h before being exposed or not exposed to MCF-7breast cancer cells. FIG. 3E, effect of CCCP on the growth of P.aeruginosa 8822 (left panel) and E. coli JM109 (right panel) cells. CCCPat the concentrations indicated were added in early to mid exponentialphase of growth and the growth rate was followed for the next 60 min atan optical density of 600 nm.

FIG. 4 depicts Pseudomonas aeruginosa 8822 strain secretesextrachromosomal DNA into the culture medium. FIG. 4A, P. aeruginosa8822 strain secretes a 15 kb extrachromosomal DNA fragment. The DNA ispurified from the culture medium filtrate by isopropanol precipitationof the nucleoprotein fractions followed by DNA purification throughQIAGEN columns (Qiagen, Inc., Valencia Calif.). FIG. 4B,Extrachromosomal DNA secretion at different time points. The DNA issecreted as early as 5 min and the secretion is enhanced at 15, 30 and60 min respectively. The DNA is efficiently secreted at all time pointsin the presence of MCF-7 human breast cancer cells. FIG. 4C, Pseudomonasaeruginosa strain 8822 secretes both azurin and the 15 kb DNA fragmentinto the culture medium filtrate in the presence of MCF-7 cancer cells.Strain 8822 was grown in the presence of the MCF-7 cancer cells and thesecretion of azurin and extrachromosomal DNA in the extracellularfraction was examined by Western blotting using anti-azurin antibody andPCR using primers for PA clone and the precipitated DNA as template. Thesecretion of azurin and the DNA is time dependent. FIG. 4D, the secretedDNA is CpG rich. The DNA is subjected to restriction endonucleasedigestion by EcoR1, HindIII, MspI and PvuI enzymes. MspI and PvuI, knownto cut CpG-rich DNA, generates a smear of DNA bands indicating thepresence of high ratio of CG sequences in the DNA.

FIG. 5 depicts the DNA sequence and amino acid translation of a highly Cand G-rich stretch of DNA in the released CpG-rich DNA. FIG. 5A, thisstretch of DNA (SEQ ID NO: 49) has no homology with any other sequencein the database and does not appear to be part of an open reading frame.FIG. 5B, comparative amino acid sequence homology of the putative azuringene present in the CpG-rich released DNA (8822) (SEQ ID NO: 63) and thecorresponding sequence from P. aeruginosa PAO1 (SEQ ID NO: 66) andNeisseria gonorrhoeae (NG) (SEQ ID NO: 64) and N. meningitidis (NM) (SEQID NO: 65).

FIG. 6 depicts the induction of NF-kB by extrachromosomal CpG-rich P.aeruginosa DNA. FIG. 6A, NF-kB induction is TLR9 dependent and requiresthe extrachromosomal CpG-rich DNA. HEK293 cells transfected withTLR9-expressing plasmid and NF-kB promoter inducible SEAP (secretedembryonic alkaline phosphatase) reporter plasmid (Invitrogen, Inc.Carlsbad, Calif.) were treated with various concentrations of CpG-rich15 kb DNA. SEAP expression following NF-kB activation was measured insupernatants of transfected cells. SEAP levels were evaluatedquantitatively using the HEK-Blue TM SEAP reporter assay kit(Invitrogen, Inc. Carlsbad, Calif.). Stimulation of HEK293 cells withCpG-rich DNA caused NF-kB induction, leading to SEAP activity in a dosedependent manner. FIG. 6B, different cancer cell lines express a broadrange of Toll-like receptors (TLRs). Expression of TLRs was analyzed byquantitative RT-PCR. The data are normalized to the expression ofcyclophilin B. FIG. 6C, effect of CpG-rich DNA treatment on cellularproliferation of MCF-7 breast cancer cells. Cells were treated withvarious concentrations of the 15 kb CpG-rich DNA (0.5, 1, 3 and 5 μg)for 12 and 24 h and cell survival was determined. MTT assay was done tomeasure the extent of live cells to account for cytotoxicity (percentcell death) as described previously (Yamada et al., 2002). To calculatepercentage cytotoxicity, the value of nontreated viable cells as 100%was used to determine the number of viable cells treated with 2.5 μgcalf thymus DNA and different concentrations of the CpG-rich DNA.

FIG. 7 is a table of the CpG rich DNA sequences released from P.aeruginosa upon contact with cancer cells.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the term “cell” includes either the singular or theplural of the term, unless specifically described as a “single cell.”

As used herein, the terms “polypeptide,” “peptide,” and “protein” areused interchangeably to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid. The terms also apply to naturally occurring aminoacid polymers. The terms “polypeptide,” “peptide,” and “protein” arealso inclusive of modifications including, but not limited to,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation. It will beappreciated that polypeptides are not always entirely linear. Forinstance, polypeptides may be branched as a result of ubiquitination andthey may be circular (with or without branching), generally as a resultof post-translation events, including natural processing event andevents brought about by human manipulation which do not occur naturally.Circular, branched and branched circular polypeptides may be synthesizedby non-translation natural process and by entirely synthetic methods aswell.

As used herein, the terms “polynucleotide,” “oligonucleotide,” “oligo,”and “DNA” are used interchangeably to refer to a polymer of nucleic acidresidues.

As used herein, the term “pharmacologic activity” means the effect of adrug or other chemical on a biological system. The effect of chemicalmay be beneficial (therapeutic) or harmful (toxic). The pure chemicalsor mixtures may be of natural origin (plant, animal, or mineral) or maybe synthetic compounds.

As used herein, the term “pathological condition” includes anatomic andphysiological deviations from the normal that constitute an impairmentof the normal state of the living animal or one of its parts, thatinterrupts or modifies the performance of the bodily functions, and is aresponse to various factors (as malnutrition, industrial hazards, orclimate), to specific infective agents (as worms, parasitic protozoa,bacteria, or viruses), to inherent defects of the organism (as geneticanomalies), or to combinations of these factors.

As used herein, the term “condition” includes anatomic and physiologicaldeviations from the normal that constitute an impairment of the normalstate of the living animal or one of its parts, that interrupts ormodifies the performance of the bodily functions.

As used herein, the term “suffering from” includes presently exhibitingthe symptoms of a pathological condition, having a pathologicalcondition even without observable symptoms, in recovery from apathological condition, or recovered from a pathological condition.

A used herein, the term “treatment” includes preventing, lowering,stopping, or reversing the progression or severity of a condition orsymptoms associated with a condition being treated. As such, the term“treatment” includes medical, therapeutic, and/or prophylacticadministration, as appropriate. Treatment may also include preventing orlessening the development of a condition, such as cancer.

As used herein, the term “inhibit cell growth” means the slowing orceasing of cell division and/or cell expansion. This term also includesthe inhibition of cell development or increases in cell death.

A “therapeutically effective amount” is an amount effective to prevent,lower, stop or reverse the development of, or to partially or totallyalleviate the existing symptoms of a particular condition for which thesubject being treated. Determination of a therapeutically effectiveamount is well within the capability of those skilled in the art.

The term “substantially pure,” as used herein, when used to modify aprotein or other cellular product of the invention, refers to, forexample, a protein isolated from the growth medium or cellular contents,in a form substantially free of, or unadulterated by, other proteinsand/or other compounds. The term “substantially pure” refers to a factorin an amount of at least about 75%, by dry weight, of isolated fraction,or at least “75% substantially pure.” More specifically, the term“substantially pure” refers to a compound of at least about 85%, by dryweight, of isolated fraction, or at least “85% substantially pure.” Mostspecifically, the term “substantially pure” refers to a compound of atleast about 95%, by dry weight, of isolated fraction, or at least “95%substantially pure.” The term “substantially pure” may also be used tomodify a synthetically-made protein or compound of the invention, where,for example, the synthetic protein is isolated from the reagents andby-products of the synthesis reaction(s).

The term “pharmaceutical grade,” as used herein, when referring to apeptide or compound of the invention, is a peptide or compound that isisolated substantially or essentially from components which normallyaccompany the material as it is found in its natural state, includingsynthesis reagents and by-products, and substantially or essentiallyisolated from components that would impair its use as a pharmaceutical.For example, a “pharmaceutical grade” peptide may be isolated from anycarcinogen. In some instances, “pharmaceutical grade” may be modified bythe intended method of administration, such as “intravenouspharmaceutical grade,” in order to specify a peptide or compound that issubstantially or essentially isolated from any substance that wouldrender the composition unsuitable for intravenous administration to apatient. For example, an “intravenous pharmaceutical grade” peptide maybe isolated from detergents, such as SDS, and anti-bacterial agents,such as azide.

The terms “isolated,” “purified” or “biologically pure” refer tomaterial which is substantially or essentially free from componentswhich normally accompany the material as it is found in its nativestate. Thus, isolated polynucleotides or peptides in accordance with theinvention preferably do not contain materials normally associated withthe polynucleotides or peptides in their in situ environment. An“isolated” region of a polynucleotides or polypeptide refers to a regionthat does not include the whole sequence of the polynucleotides orpolypeptide from which the region was derived. An “isolated” nucleicacid, protein, or respective fragment thereof has been substantiallyremoved from its in vivo environment so that it may be manipulated bythe skilled artisan, such as but not limited to, nucleotide sequencing,restriction digestion, site-directed mutagenesis, and subcloning intoexpression vectors for a nucleic acid fragment as well as obtaining theprotein or protein fragment in substantially pure quantities.

The term “variant” as used herein with respect to a peptide, refers toamino acid sequence variants which may have amino acids replaced,deleted, or inserted as compared to the wild-type polypeptide. Variantsmay be truncations of the wild-type peptide. A “deletion” is the removalof one or more amino acids from within the polypeptide, while a“truncation” is the removal of one or more amino acids from one or bothends of the polypeptide. Thus, a variant peptide may be made bymanipulation of genes encoding the polypeptide. A variant may be made byaltering the basic composition or characteristics of the polypeptide,but not at least some of its pharmacologic activities. For example, a“variant” of azurin can be a mutated azurin that retains its ability toinhibit the development of premalignant mammalian cells. In some cases,a variant peptide is synthesized with non-natural amino acids, such asε-(3, 5-dinitrobenzoyl)-Lys residues. Ghadiri & Fernholz, J. Am. Chem.Soc., 112:9633-9635 (1990). In some embodiments, the variant has notmore than 20 amino acids replaced, deleted or inserted compared towild-type peptide or part thereof. In some embodiments, the variant hasnot more than 15 amino acids replaced, deleted or inserted compared towild-type peptide or part thereof. In some embodiments, the variant hasnot more than 10 amino acids replaced, deleted or inserted compared towild-type peptide or part thereof. In some embodiments, the variant hasnot more than 6 amino acids replaced, deleted or inserted compared towild-type peptide or part thereof. In some embodiments, the variant hasnot more than 5 amino acids replaced, deleted or inserted compared towild-type peptide or part thereof. In some embodiments, the variant hasnot more than 3 amino acids replaced, deleted or inserted compared towild-type peptide or part thereof.

The term “amino acid,” as used herein, means an amino acid moiety thatcomprises any naturally-occurring or non-naturally occurring orsynthetic amino acid residue, i.e., any moiety comprising at least onecarboxyl and at least one amino residue directly linked by one, twothree or more carbon atoms, typically one (a) carbon atom.

The term “derivative” as used herein with respect to a peptide refers toa peptide that is derived from the subject peptide. A derivationincludes chemical modifications of the peptide such that the peptidestill retains some of its fundamental activities. For example, a“derivative” of azurin can, for example, be a chemically modified azurinthat retains its ability to inhibit angiogenesis in mammalian cells.Chemical modifications of interest include, but are not limited to,amidation, acetylation, sulfation, polyethylene glycol (PEG)modification, phosphorylation or glycosylation of the peptide. Inaddition, a derivative peptide may be a fusion of a polypeptide orfragment thereof to a chemical compound, such as but not limited to,another peptide, drug molecule or other therapeutic or pharmaceuticalagent or a detectable probe.

The term “percent (%) nucleotide sequence identity” is defined as thepercentage of nucleotides in a polynucleotide that are identical withnucleotides in a candidate sequence when the two sequences are aligned.To determine % nucleotide identity, sequences are aligned and ifnecessary, gaps are introduced to achieve the maximum % sequenceidentity. Nucleotide sequence alignment procedures to determine percentidentity are well known to those of skill in the art. Often publiclyavailable computer software such as BLAST, BLAST2, ALIGN2 or Megalign(DNASTAR) software is used to align nucleotide sequences. In a specificembodiment, Blastn (available from the National Center for BiotechnologyInformation, Bethesda Md.) may be used using the default parameters oflow complexity filter, expect 10, and word size 11.

The term “host cell” includes an individual cell or cell culture whichcan be or has been a recipient of any recombinant vector(s) or isolatedpolynucleotide of the invention. Host cells include progeny of a singlehost cell, and the progeny may not necessarily be completely identical(in morphology or in total DNA complement) to the original parent celldue to natural, accidental, or deliberate mutation and/or change. A hostcell includes cells transfected or infected in vivo or in vitro with arecombinant vector or a polynucleotide of the invention. A host cellwhich comprises a recombinant vector of the invention is a “recombinanthost cell”.

The term “transformation” is used interchangeably with “geneticmodification” and refers to a permanent or transient genetic changeinduced in a cell following introduction of new DNA (i.e., DNA exogenousto the cell). Genetic change (“modification”) can be accomplished eitherby incorporation of the new DNA into the genome of the host cell, or bytransient or stable maintenance of the new DNA as an episomal element.

When amino acid sequences are aligned, the % amino acid sequenceidentity of a given amino acid sequence A to, with, or against a givenamino acid sequence B (which can alternatively be phrased as a givenamino acid sequence A that has or comprises a certain % amino acidsequence identity to, with, or against a given amino acid sequence B)can be calculated as:% amino acid sequence identity=X/Y*100

where

-   -   X is the number of amino acid residues scored as identical        matches by the sequence alignment program's or algorithm's        alignment of A and B and    -   Y is the total number of amino acid residues in B.

If the length of amino acid sequence A is not equal to the length ofamino acid sequence B, the % amino acid sequence identity of A to B willnot equal the % amino acid sequence identity of B to A. When comparinglonger sequences to shorter sequences, the shorter sequence will be the“B” sequence. For example, when comparing truncated peptides to thecorresponding wild-type polypeptide, the truncated peptide will be the“B” sequence.

When nucleotide sequences are aligned, the % nucleotide sequenceidentity of a given nucleotide sequence A to, with, or against a givennucleotide sequence B (which can alternatively be phrased as a givennucleotide sequence A that has or comprises a certain % nucleotidesequence identity to, with, or against a given nucleotide sequence B)can be calculated as:% nucleotide sequence identity=X/Y*100

where

-   -   X is the number of nucleotide residues scored as identical        matches by the sequence alignment program's or algorithm's        alignment of A and B and    -   Y is the total number of nucleotide residues in B.

If the length of nucleotide sequence A is not equal to the length ofnucleotide sequence B, the % nucleotide sequence identity of A to B willnot equal the % nucleotide sequence identity of B to A. When comparinglonger sequences to shorter sequences, the shorter sequence will be the“B” sequence. For example, when comparing a region of a polynucleotideto the corresponding full-length polynucleotide, the region of thepolynucleotide will be the “B” sequence.

General

The present invention provides compositions comprising isolatedpolynucleotides comprising CpG rich DNA from Pseudomonas aeruginosa. Theinvention also provides pharmaceutical compositions comprising CpG richDNA from Pseudomonas aeruginosa, and optionally at least one cupredoxinpeptide, which may be advantageously used to treat patients,specifically patients suffering from a condition, and specificallycancer, or to prevent cancer. The invention further provides methods totreat patients, specifically patients suffering from conditions,specifically cancer, or to prevent cancer, comprising administering theCpG rich DNA from P. aeruginosa optionally in combination with acupredoxin peptide. The present also provides cells that are induced toexpress specific proteins when in contact with cancer cells using theazurin gene from P. aeruginosa. These cells maybe used in methods totreat a patient, specifically suffering from a condition, specificallycancer, or to prevent cancer, or to diagnose cancer in a patient.

Relating to a first aspect of the invention, it was previously knownthat a redox protein elaborated by Pseudomonas aerugisnosa, thecupredoxin azurin, selectively enters J774 lung cancer cells but notnormal cells, and induces apoptosis. Zaborina et al., Microbiology146:2521-2530 (2000). Azurin can also selectively enter and kill humanmelanoma UISO-Mel-2 or human breast cancer MCF-7 cells. Yamada et al.,PNAS 99:14098-14103 (2002); Punj et al., Oncogene 23:2367-2378 (2004).Azurin from P. aeruginosa preferentially enters J774 murine reticulumcell sarcoma cells, forms a complex with and stabilizes the tumorsuppressor protein p53, enhances the intracellular concentration of p53,and induces apoptosis. Yamada et al., Infection and Immunity70:7054-7062 (2002). Detailed studies of various domains of the azurinmolecule showed that amino acids 50-77 (p28) (SEQ ID NO: 2) representeda protein transduction domain (PTD) critical for internalization andsubsequent apoptotic activity. Yamada et al., Cell. Microbial.7:1418-1431 (2005).

It is now known that Pseudomonas aeruginosa also releases specific DNAsequences into the extracellular medium, in a manner enhanced by thepresence of cancer cells. See Example 5. This DNA is released from P.aeruginosa as soon as 5 minutes after contact with cancer cells,suggesting that the released DNA is extrachromosomal in origin. SeeExample 6. Digestion of the released DNA with restriction enzymesindicates that the DNA is rich in G+C nucleotides, and thus is “CpG-richDNA.” See Example 7. Among the DNA sequences found among the releasedDNA is a sequence, SEQ ID NO: 26, that is CpG rich and is 95% identicalto the nucleotide sequence from the azurin gene from Neisseria, laz. SeeExample 8. Finally, this P. aeruginosa CpG rich DNA preparation is nowknown to have anti-tumor properties, as documented by its ability toactivate NF-kB in a TLR9-dependent manner. See Example 9.

It is contemplated that the P. aeruginosa CpG rich DNA may beadministered with cupredoxins to treat patients, specifically sufferingfrom a condition, specifically cancer, or to prevent cancer.Specifically, it is contemplated that the polynucleotide released fromP. aeruginosa with the sequence that is very similar to the Neisseriallaz gene, SEQ ID NO: 26, may be coadministered with a cupredoxin, suchas azurin from Pseudomonas aeruginosa, and/or the 50-77 residue regionof azurin (p28), to improve the efficaciousness of cupredoxin alone ontreating patients with cancer, AIDS, malaria, inappropriate angiogenesisor at risk of developing cancer. While not limiting the function of thistreatment method to any one means, it is contemplated that the CpG richDNA released by P. aeruginosa will decrease the degree by which theimmune system of the patient attacks the co-administered cupredoxinpeptide. It is contemplated that P. aeruginosa has evolved as a parasiteon mammalian species, and that tumor growth on the mammalian speciesinhibits the growth of P. aeruginosa. Further, it is thought that P.aeruginosa actively secretes azurin on exposure to cancer cells as aweapon against cancer. Since azurin can be targeted by the host immunesystem for antibody formation, it is contemplated that P. aeruginosastrain 8822 has also evolved CpG-rich DNA by to be released uponexposure to cancer cells so that it can be used as a decoy to divert theattention of the immune system from targeting azurin. Because the CpGrich DNA is thought to protect an optionally co-administered cupredoxin,it will be effective in improving the efficaciousness of any treatmentmethod relating to administering a cupredoxin, regardless of the diseaseor condition being treated. It is further contemplated that the CpG richDNA is useful in protecting any co-administered chemical compound fromtargeting by the immune system.

Relating to the second aspect of the invention, it was previously knownthat azurin from Pseudomonas aeruginosa is a periplasmic protein whichis secreted in the growth medium at the late stage of growth. Zaborinaet al., Microbiology 146:2521-2530 (2000). Its extracellular release isdependent on quorum sensing at high cell density and modulated by GacAor the two small RNA products RsmY and RsmZ. Kay et al., J. Bacteriol.188:6026-6033 (2006).

It is now known that the presence of human cancer cells induces P.aeruginosa to release azurin into the extracellular medium. In thepresence of cancer cell lines MCF-7 and Mel-2, P. aeruginosa releasedazurin into the medium within 20 to 30 min of exposure to the cancercells, but no secretion was observed in the absence of the cancer cells.See Example 1. Further, this release of azurin was not due to celllysis, and required the actual presence of the cancer cells, in contrastto diffusible factors from the cancer cells. See Example 2. Further, itis now known that cancer cells will also induce the release of azurin

from E. coli harboring the P. aeruginosa azurin gene. See Example 3.Finally, energy is not required to release azurin from P. aeruginosa orE. coli. See Example 4.

Live cells of M. bovis are widely used today in the treatment forsuperficial bladder cancer, and the use of attenuated bacteria in thepotential therapy against cancer is attracting wide attention.Chakrabarty, J. Bacterial. 185:2683-2686 (2003); Minton, Nature Rev.Microbiol. 1:237-243 (2003); Dang et al., Cancer Biol. Therap.,3:326-337 (2004); Vassaux et al., J. Pathol. 208:290-298 (2006). It iscontemplated that the azurin gene from P. aeruginosa can be used in P.aeruginosa or in another cell, such as E. coli, to express azurin or aheterologous protein when in contact with cancer cells. A cell harboringthe azurin gene expressing azurin or a heterologous protein will beuseful to treat cancer by delivering the expressed protein to the siteof the cancer cells, or to provide a means by which the location ofcancer could be diagnosed by the expression of the protein.

Compositions of the Invention

The invention provides for isolated polynucleotides that are releasedfrom P. aeruginosa and that are useful for treating patients,specifically suffering from conditions, and specifically cancer. In someembodiments, the isolated polynucleotides are CpG rich DNA. In someembodiments, the isolated polynucleotides are those released by P.aeruginosa upon contact with cancer cells and prepared according to theprocedure provided in Example 5. In other embodiments, the isolated CpGrich DNA may comprise one or more isolated polynucleotides with thesequences provided in SEQ ID NOS: 26-62. In specific embodiments, theisolated CpG rich DNA comprises isolated polynucleotides with thesequence in SEQ ID NO: 26. In other specific embodiments, the CpG richDNA consists of an isolated polynucleotide with the sequence in SEQ IDNOS: 26-62.

The invention provides compositions comprising isolated CpG rich DNAfrom P. aeruginosa, optionally with at least one isolated cupredoxinpeptide. In some embodiments, these compositions also comprise apharmaceutically acceptable carrier. In specific embodiments, thecomposition is designed for a particular mode of administration, forexample, but not limited to, oral, intraperitoneal, or intravenous. Suchcompositions may be hydrated in water, or may be dried (such as bylyophilization) for later hydration. Such compositions may be insolvents other than water, such as but not limited to, alcohol.

The CpG rich DNA may be in the form of stabilized DNA. In oneembodiment, the DNA can be stabilized by condensing the DNA viatreatment with calcium chloride in 20% (v/v) tert-butanol, so as to formsmall (about 50 nm) toroids as well as larger (about 300 nm) rods andspheres i.e. condensed particles of DNA. The condensed particles retaina negative surface charge, indicating sub-stoichiometric concentrationsof calcium. More particularly, in one embodiment, purified deionized DNAat a concentration of about 0.1 μg/mL to about 1 mg/mL can be dissolvedin an aqueous solution of t-butanol ranging in concentration from about17% to about 25% (v/v). An appropriate divalent cation consisting ofCa⁺², Mg⁺², or Zn²⁺ at concentrations of about 0.2 mM to about 2 mM canthen be added to the t-butanol cosolvent solution to condense the DNA.In one embodiment, the stoichiometric ratio of anionic phosphates of theDNA backbone to divalent cations is between (anions/cations) about 0.1and about 1.0, with about 0.3 being a most preferred ratio. The solutioncan then be allowed to equilibrate, for about 45 minutes to permitthermodynamic equilibrium condensation to be attained. The rods,toroids, and spherical DNA particulates will fall in the size range ofabout 20 to about 500 nm. The solution containing the condensed DNA canthen be transferred to downstream process unit operations, such as,without limitation, sterile filtration through 0.22 um filters,spray-drying, or lyophilization.

The sterile filtered condensed DNA can then be processed bylyophilization or spray-drying to obtain stable pharmaceutical dosageforms. Prior to lyophilization the DNA can be combined with bulkingagents such as sucrose, mannitol, trehalose, lactose, or other commonbulking agents. The t-butanol solution freezes and forms a single phaseamenable to lyophilization due to the sublimation properties oft-butanol. In the dried lyophilized cake the DNA toroids and rods remainintact. Upon reconstitution the lyophile cake may rapidly dissolve andthe DNA becomes completely solubilized into uncondensed native plasmidDNA ready for dosing with the only traces of the original process beingthe cations and any added bulking agents. It is substantially free oft-butanol at this stage of the process.

An alternate approach to processing the DNA is to utilize spray drying.Spray drying may involve three fundamental unit processes: liquidatomization, gas-droplet mixing, and drying from liquid droplets.Atomization is accomplished usually by one of three atomizing devices:high-pressure nozzles, two-fluid nozzles, and high-speed centrifugaldisks. With these atomizers, thin solutions may be dispersed intodroplets as small as about 2 μm. The largest drop sizes rarely exceedabout 500 μm (35 mesh). Because of the large total drying surface andsmall droplet sizes created, the actual drying time in a spray dryer istypically not more than about 30 seconds.

One of the principal advantages of spray drying is the production of aspherical particle, which is usually not obtainable by any other dryingmethods. The spherical particle may be solid or hollow, depending on thematerial, the feed condition, and the drying conditions. Because of thehigh heat-transfer rates to the drops, the liquid at the center of theparticle vaporizes, causing the outer shell to expand and form a hollowsphere.

The dried DNA particles can be used as a powdered form of the DNA readyto be reconstituted into a hydrating solution for parenteraladministration including, but not limited to intravenous, intramuscularand intraperitoneal administration. The reconstituted DNA can also beadministered subcutaneously and intraocularly. The reconstituted DNA canalso be administered by aerosol means and can be delivered in a driedparticulate form directly in a powder by aerosol or other inhalatoryadministration means. The composition of powdered DNA for dosing issubstantially free of the solvents used to modify its structure.

The isolated polynucleotide may be similar but not identical to one ormore of the DNA sequences found in the DNA released by P. aeruginosaupon contact with cancer cells. In one embodiment, the isolatedpolynucleotide may have more than about 80% nucleotide sequence identitywith one or more of SEQ ID NOS: 26-62. In another embodiment, theisolated polynucleotide may have more than about 90% nucleotide sequenceidentity with one or more of SEQ ID NOS: 26-62. In another embodiment,the isolated polynucleotide may have more than about 95% nucleotidesequence identity with one or more of SEQ ID NOS: 26-62.

The compositions of the invention may further comprise a cupredoxinpeptide which has anti-tumor activity or other pharmacologic activity ofinterest. Cupredoxin peptides may be cupredoxins, variants, derivativesor structural equivalents thereof, as provided in U.S. Pat. No.7,084,105, U.S. Patent Application Publication No. 20060040269,published Feb. 23, 2006, now U.S. Pat. No. 7,491,394; U.S. PatentApplication Publication No. 20060149037, published Jul. 6, 2006, nowU.S. Pat. No. 7,691,383; U.S. Patent Application Publication No.20080213185, published Sep. 4, 2008, now U.S. Pat. No. 7,807,183; U.S.Patent Application Publication No. 20080089878, published Apr. 17, 2008,now U.S. Pat. No. 7,381,701; U.S. Patent Application Publication No.20080103087, published May 1, 2008, now U.S. Pat. No. 7,556,810; U.S.Patent Application Publication No. 20060251669, now U.S. Pat. No.7,338,766; U.S. Patent Application Publication No. 20060251639,published Nov. 9, 2006, now U.S. Pat. No. 7,301,010; U.S. PatentApplication Publication No. 20100267608, published Oct. 21, 2010; andU.S. Patent Application Publication No. 20080139471, published Jun. 12,2008, now U.S. Pat. No. 7,618,939, each of which is incorporated hereinby reference. In specific embodiments, the cupredoxin peptide may haveat least one pharmacologic activity of a cupredoxin. Specificpharmacologic activities of interest include (1) entering a mammaliancancer cell, (2) not entering non-cancerous mammalian cells, (3)entering pre-malignant mammalian cells, (4) killing mammalian cancercells, (5) killing pre-malignant mammalian cells, (6) inhibiting thegrowth of a mammalian cancer cell, (7) inhibiting HIV-1 infection, (8)inhibiting parasitemia of malaria-infected red blood cells, (9)interfering with the ephrin signaling system, (10) inhibitingangiogenesis and (11) inhibiting the development of premalignantlesions.

The cupredoxin peptide may be a full length wild-type cupredoxin, orvariants, derivatives or structural equivalents of cupredoxin thatexhibit one or more of the pharmacologic activities in mammalian cells,tissues and/or animals. In some embodiments, the cupredoxin peptide isisolated. In some embodiments, the cupredoxin peptide is substantiallypure or pharmaceutical grade. In other embodiments, the cupredoxinpeptide is in a composition that comprises, or consists essentially of,the peptide. In another specific embodiment, the cupredoxin peptide doesnot raise an immune response in a mammal, and more specifically a human.In some embodiments, the cupredoxin peptide is less than a full lengthcupredoxin, and retains some of the pharmacologic activities of thecupredoxins.

Because of the high structural homology between the cupredoxins, it iscontemplated that cupredoxin peptides will have the same pharmacologicactivity as p28 (SEQ ID NO: 2). In some embodiments, the cupredoxinpeptide is, but is not limited to, azurin, pseudoazurin, plastocyanin,rusticyanin, auracyanin or Laz. In particularly specific embodiments,the azurin is derived from Pseudomonas aeruginosa, Alcaligenes faecalis,Achromobacter xylosoxidans ssp. denitrificans I, Bordetellabronchiseptica, Methylomonas sp., Neisseria meningitidis, Neisseriagonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis, Xylellafastidiosa or Vibrio parahaemolyticus. In a very specific embodiment,the azurin is from Pseudomonas aeruginosa. In other specificembodiments, the cupredoxin peptide comprises an amino acid sequencethat is SEQ ID NO: 1, 3-19.

The cupredoxin peptides may be amino acid sequence variants which haveamino acids replaced, deleted, or inserted as compared to the wild-typecupredoxin. The cupredoxin peptides may be truncations of the wild-typecupredoxin. In some embodiments, the cupredoxin peptide comprises aregion of a cupredoxin that is less than the full length wild-typepolypeptide. In some embodiments, the cupredoxin peptide comprises morethan about 10 residues, more than about 15 residues or more than about20 residues of a truncated cupredoxin. In some embodiments, thecupredoxin peptide comprises not more than about 100 residues, not morethan about 50 residues, not more than about 40 residues, not more thanabout 30 residues or not more than about 20 residues of a truncatedcupredoxin. In some embodiments, a cupredoxin has to the cupredoxinpeptide, and more specifically SEQ ID NOS: 1-19, at least about 70%amino acid sequence identity, at least about 80% amino acid sequenceidentity, at least about 90% amino acid sequence identity, at leastabout 95% amino acid sequence identity or at least about 99% amino acidsequence identity.

In specific embodiments, the cupredoxin peptide comprises P. aeruginosaazurin residues 50-77, azurin residues 50-67, or azurin residues 36-88.In other embodiments, the cupredoxin peptide consists of P. aeruginosaazurin residues 50-77, azurin residues 50-67, or azurin residues 36-88.In other specific embodiments, the cupredoxin peptide consists of theequivalent residues of a cupredoxin other than azurin. It is alsocontemplated that other cupredoxin peptides can be designed that have asimilar activity to azurin residues 50-77, azurin residues 50-67, orazurin residues 36-88. To do this, the subject cupredoxin amino acidsequence will be aligned to the Pseudomonas aeruginosa azurin sequenceusing BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR), the relevant residueslocated on the P. aeruginosa azurin amino acid sequence, and theequivalent residues found on the subject cupredoxin sequence, and theequivalent cupredoxin peptide thus designed.

In one embodiment of the invention, the cupredoxin peptide contains atleast amino acids 57 to 89 of auracyanin B of Chloroflexus aurantiacus(SEQ ID NO: 20). In another embodiment of the invention, the cupredoxinpeptide contains at least amino acids 51-77 of Bordetella pertussisazurin (SEQ ID NO: 21). In another embodiment of the invention, thecupredoxin peptide contains at least amino acids 51-77 of Pseudomonassyringae azurin (SEQ ID NO: 23). In another embodiment of the invention,the cupredoxin peptide contains at least amino acids 89-115 of Neisseriameningitidis Laz (SEQ ID NO: 22). In another embodiment of theinvention, the cupredoxin peptide contains at least amino acids 52-78 ofVibrio parahaemolyticus azurin (SEQ ID NO: 24). In another embodiment ofthe invention, the cupredoxin peptide contains at least amino acids51-77 of Bordetella bronchiseptica azurin (SEQ ID NO: 25).

The cupredoxin peptides also include peptides made with synthetic aminoacids not naturally occurring. For example, non-naturally occurringamino acids may be integrated into the cupredoxin peptide to extend oroptimize the half-life of the composition in the bloodstream. Suchvariants include, but are not limited to, D,L-peptides (diastereomer),(for example Futaki et al., J. Biol. Chem. 276(8):5836-40 (2001); Papoet al., Cancer Res. 64(16):5779-86 (2004); Miller et al, Biochem.Pharmacol. 36(1):169-76, (1987); peptides containing unusual amino acids(for example Lee et al., J. Pept. Res. 63(2):69-84 (2004)),olefin-containing non-natural amino acid followed by hydrocarbonstapling (for example Schafmeister et al., J. Am. Chem. Soc.122:5891-5892 (2000); Walenski et al., Science 305:1466-1470 (2004)),and peptides comprising ε-(3,5-dinitrobenzoyl)-Lys residues.

In other embodiments, the cupredoxin peptide is a derivative of acupredoxin. The derivatives of cupredoxin are chemical modifications ofthe peptide such that the peptide still retains some of its fundamentalactivities. For example, a “derivative” of azurin can be a chemicallymodified azurin that retains its ability to kill mammalian cancer cells,tissues or animals. Chemical modifications of interest include, but arenot limited to, hydrocarbon stabling, amidation, acetylation, sulfation,polyethylene glycol (PEG) modification, phosphorylation andglycosylation of the peptide. In addition, a cupredoxin peptide may be afusion of a cupredoxin peptide to a chemical compound, such as but notlimited to, another peptide, drug molecule or other therapeutic orpharmaceutical agent or a detectable probe. Derivatives of interestinclude chemical modifications by which the half-life in the bloodstreamof the peptides and compositions of the invention can be extended oroptimized, such as by several methods well known to those in the art,including but not limited to, circularized peptides (for example Monk etal., BioDrugs 19(4):261-78, (2005); DeFreest et al., J. Pept. Res.63(5):409-19 (2004)), N- and C-terminal modifications (for exampleLabrie et al., Clin. Invest. Med. 13(5):275-8, (1990)), andolefin-containing non-natural amino acid followed by hydrocarbonstapling (for example Schafmeister et al., J. Am. Chem. Soc.122:5891-5892 (2000); Walenski et al., Science 305:1466-1470 (2004)).

In addition, a cupredoxin peptide may be a fusion of a cupredoxinpeptide to a cargo, such as a chemical compound, including but notlimited to, another peptide, drug molecule or other therapeutic orpharmaceutical agent or a detectable probe. In one embodiment, adetectable substance, for example, a fluorescent substance, such asgreen fluorescent protein; a luminescent substance; an enzyme, such asβ-galactosidase; or a radiolabelled or biotinylated protein is deliveredto confer a detectable phenotype to a cell. Similarly, microparticles ornanoparticles labeled with a detectable substance, for example, afluorescent substance, can be delivered. One example of suitablenanoparticles is found in U.S. Pat. No. 6,383,500, issued May 7, 2002,which is expressly incorporated by reference herein. Many suchdetectable substances are known to those skilled in the art.

In some embodiments, the cargo compound is a detectable substance thatis suitable for X-ray computed tomography, magnetic resonance imaging,ultrasound imaging or radionuclide scintigraphy. In these embodiments,the cargo compound is administered to the patient for purposes ofdiagnosis. A contrast agent is administered as a cargo compound toenhance the image obtained by X-ray CT, MRI and ultrasound. Theadministration of a radionuclide cargo compound that is targeted totumor tissue via the cupredoxin entry domain can be used forradionuclide scintigraphy. In some embodiments, the cupredoxin entrydomain may contain the radionucleotide with or without a cargo compound.In other embodiments, the cargo compound is a gamma ray or positronemitting radioisotope, a magnetic resonance imaging contrast agent, anX-ray contrast agent, or an ultrasound contrast agent.

Ultrasound contrast agents suitable for use as cargo compounds include,but are not limited to, a microbubble of a biocompatible gas, a liquidcarrier, and a surfactant microsphere, further comprising an optionallinking moiety, L., between the targeting moieties and the microbubble.In this context, the term liquid carrier means aqueous solution and theterm surfactant means any amphiphilic material which produces areduction in interfacial tension in a solution. A list of suitablesurfactants for forming surfactant microspheres is disclosed inEP0727225A2, herein expressly incorporated by reference. The termsurfactant microsphere includes nanospheres, liposomes, vesicles and thelike. The biocompatible gas can be air, or a fluorocarbon, such as aC₃-C₅ perfluoroalkane, which provides the difference in echogenicity andthus the contrast in ultrasound imaging. The gas is encapsulated orcontained in the microsphere to which is attached the cupredoxin entrydomain, optionally via a linking group. The attachment can be covalent,ionic or by van der Waals forces. Specific examples of such contrastagents include lipid encapsulated perfluorocarbons with a plurality oftumor neovasculature receptor binding peptides, polypeptides orpeptidomimetics.

X-ray contrast agents suitable for use as cargo compounds include, butare not limited to, one or more X-ray absorbing or “heavy” atoms ofatomic number 20 or greater, further comprising an optional linkingmoiety, L., between the cupredoxin entry domain and the X-ray absorbingatoms. The frequently used heavy atom in X-ray contrast agents isiodine. Recently, X-ray contrast agents comprised of metal chelates(e.g., U.S. Pat. No. 5,417,959) and polychelates comprised of aplurality of metal ions (e.g., U.S. Pat. No. 5,679,810) have beendisclosed. More recently, multinuclear cluster complexes have beendisclosed as X-ray contrast agents (e.g., U.S. Pat. No. 5,804,161, PCTWO91/14460, and PCT WO 92/17215).

MRI contrast agents suitable for use as cargo compounds include, but arenot limited to, one or more paramagnetic metal ions, further comprisingan optional linking moiety, L_(n), between the cupredoxin entry domainand the paramagnetic metal ions. The paramagnetic metal ions are presentin the form of metal complexes or metal oxide particles. U.S. Pat. Nos.5,412,148, and 5,760,191, describe examples of chelators forparamagnetic metal ions for use in MRI contrast agents. U.S. Pat. No.5,801,228, U.S. Pat. No. 5,567,411, and U.S. Pat. No. 5,281,704,describe examples of polychelants useful for complexing more than oneparamagnetic metal ion for use in MRI contrast agents. U.S. Pat. No.5,520,904, describes particulate compositions comprised of paramagneticmetal ions for use as MRI contrast agents.

In another embodiment, a cargo compound is delivered to kill or retardcell cycle progression in a cell, such as a cancer cell. Such a cancercell can be, for example, an osteosarcoma cell, lung carcinoma cell,colon carcinoma cell, lymphoma cell, leukemia cell, soft tissue sarcomacell or breast, liver, bladder or prostate carcinoma cell. For example,the cargo compound can be a cell cycle control protein, such as p53; acyclin-dependent kinase inhibitor, such as p16, p21 or p2′7; a suicideprotein such as thymidine kinase or nitroreductase; a cytokine or otherimmunomodulatory protein such as interleukin 1, interleukin 2 orgranulocyte-macrophage colony stimulating factor (GM-CSF); or a toxin,such as Pseudomonas aeruginosa exotoxin A. In other embodiments, abiologically active fragment of one of the above classes of compounds isdelivered.

In yet another embodiment, the cargo compound is a nucleic acid codingfor one of the above classes of compounds. In yet another embodiment,the cargo compound is a drug used to treat cancer. Such drugs include,for example, 5-fluorouracil; Interferon α; Methotrexate; Tamoxifen; andVincristine. The above examples are provided for illustration only, manyother such compounds are known to those skilled in the art.

Cargo compounds suitable for treating cancer include, but not limitedto, alkylating agents such as nitrogen mustards, alkyl sulfonates,nitrosoureas, ethylenimines, and triazenes; antimetabolites such asfolate antagonists, purine analogues, and pyrimidine analogues;antibiotics such as anthracyclines, bleomycins, mitomycin, dactinomycin,and plicamycin; enzymes such as L-asparaginase; farnesyl-proteintransferase inhibitors; 5.alpha.-reductase inhibitors; inhibitors of17β-hydroxysteroid dehydrogenase type 3; hormonal agents such asglucocorticoids, estrogens/antiestrogens, androgens/antiandrogens,progestins, and luteinizing hormone-releasing hormone antagonists,octreotide acetate; microtubule-disruptor agents, such as ecteinascidinsor their analogs and derivatives; microtubule-stabilizing agents such astaxanes, for example, paclitaxel, docetaxel, and their analogs, andepothilones, such as epothilones A-F and their analogs; plant-derivedproducts, such as vinca alkaloids, epipodophyllotoxins, taxanes; andtopiosomerase inhibitors; prenyl-protein transferase inhibitors; andmiscellaneous agents such as hydroxyurea, procarbazine, mitotane,hexamethylmelamine, platinum coordination complexes such as cisplatinand carboplatin; and other agents used as anti-cancer and cytotoxicagents such as biological response modifiers, growth factors; immunemodulators and monoclonal antibodies.

Representative examples of these classes of anti-cancer and cytotoxicagents include but are not limited to mechlorethamine hydrochloride,cyclophosphamide, chlorambucil, melphalan, ifosfamide, busulfan,carmustin, lomustine, semustine, streptozocin, thiotepa, dacarbazine,methotrexate, thioguanine, mercaptopurine, fludarabine, pentastatin,cladribin, cytarabine, fluorouracil, doxorubicin hydrochloride,daunorubicin, idarubicin, bleomycin sulfate, mitomycin C, actinomycin D,safracins, saframycins, quinocarcins, discodermolides, vincristine,vinblastine, vinorelbine tartrate, etoposide, etoposide phosphate,teniposide, paclitaxel, tamoxifen, estramustine, estramustine phosphatesodium, flutamide, buserelin, leuprolide, pteridines, diyneses,levamisole, aflacon, interferon, interleukins, aldesleukin, filgrastim,sargramostim, rituximab, BCG, tretinoin, irinotecan hydrochloride,betamethosone, gemcitabine hydrochloride, altretamine, and topoteca andany analogs or derivatives thereof.

Preferred members of these classes include, but are not limited to,paclitaxel, cisplatin, carboplatin, doxorubicin, carminomycin,daunorubicin, aminopterin, methotrexate, methopterin, mitomycin C,ecteinascidin 743, or pofiromycin, 5-fluorouracil, 6-mercaptopurine,gemcitabine, cytosine arabinoside, podophyllotoxin or podophyllotoxinderivatives such as etoposide, etoposide phosphate or teniposide,melphalan, vinblastine, vincristine, leurosidine, vindesine andleurosine.

Examples of anticancer and other cytotoxic agents useful as cargocompounds include the following: epothilone derivatives as found inGerman Patent No. 4138042.8; WO 97/19086, WO 98/22461, WO 98/25929, WO98/38192, WO 99/01124, WO 99/02224, WO 99/02514, WO 99/03848, WO99/07692, WO 99/27890, WO 99/28324, WO 99/43653, WO 99/54330, WO99/54318, WO 99/54319, WO 99/65913, WO 99/67252, WO 99/67253 and WO00/00485; cyclin dependent kinase inhibitors as found in WO 99/24416(see also U.S. Pat. No. 6,040,321); and prenyl-protein transferaseinhibitors as found in WO 97/30992 and WO 98/54966; and agents such asthose described generically and specifically in U.S. Pat. No. 6,011,029(the compounds of which U.S. patent can be employed together with anyNHR modulators (including, but not limited to, those of presentinvention) such as AR modulators, ER modulators, with LHRH modulators,or with surgical castration, especially in the treatment of cancer).

The above other therapeutic agents, when employed as cargo compoundswith the compounds of the present invention, may be used, for example,in those amounts indicated in the Physicians' Desk Reference (PDR) or asotherwise determined by one of ordinary skill in the art.

In another embodiment, the cupredoxin peptide is a structural equivalentof a cupredoxin. Examples of studies that determine significantstructural homology between cupredoxins and other proteins include Tothet al. (Developmental Cell 1:82-92 (2001)). Specifically, significantstructural homology between a cupredoxin and the structural equivalentis determined by using the VAST algorithm. Gibrat et al., Curr OpinStruct Biol 6:377-385 (1996); Madej et al., Proteins 23:356-3690 (1995).In specific embodiments, the VAST p value from a structural comparisonof a cupredoxin to the cupredoxin peptide is less than about 10⁻³, lessthan about 10⁻⁵, or less than about 10⁻⁷. In other embodiments,significant structural homology between a cupredoxin and the cupredoxinpeptide is determined by using the DALI algorithm. Holm & Sander, J.Mol. Biol. 233:123-138 (1993). In specific embodiments, the DALI Z scorefor a pairwise structural comparison is at least about 3.5, at leastabout 7.0, or at least about 10.0.

It is contemplated that the cupredoxin peptides may be more than one ofa variant, derivative and/or structural equivalent of a cupredoxin. Forexample, the cupredoxin peptides may be a truncation of azurin that hasbeen PEGylated, thus making it both a variant and a derivative. In oneembodiment, the cupredoxin peptides are synthesized withα,α-disubstituted non-natural amino acids containing olefin-bearingtethers, followed by an all-hydrocarbon “staple” by ruthenium catalyzedolefin metathesis. Scharmeister et al., J. Am. Chem. Soc. 122:5891-5892(2000); Walensky et al., Science 305:1466-1470 (2004). Additionally,cupredoxin peptides that are structural equivalents of azurin may befused to other peptides, thus making a peptide that is both a structuralequivalent and a derivative. These examples are merely to illustrate andnot to limit the invention. Cupredoxin peptides may or may not bindcopper.

In some embodiments, the cupredoxin peptide has some of the functionalcharacteristics of the P. aeruginosa azurin, and specifically p28. In aspecific embodiment, the cupredoxin peptides may inhibit angiogenesis inmammalian cells, tissues or animals, and specifically but not limitedto, HUVECs. The cupredoxin peptides may have the ability to inhibit thegrowth of mammalian cancer cells, and specifically but not limited to,melanoma, breast, pancreas, glioblastoma, astrocytoma, or lung cancercells. The cupredoxin peptide may have the ability to inhibit thedevelopment of premalignant mammalian cells. The cupredoxin peptide mayhave the ability to enter mammalian cancer cells as compared toequivalent non-cancer cells, specifically, but not limited to, melanoma,breast, pancreas, glioblastoma, astrocytoma, or lung cancer cellsInhibition of angiogenesis or growth of cancer cells is any decrease, orlessening of the rate of increase, of that activity that isstatistically significant as compared to control treatments. The entryinto cells is any rate of entry into the cells that is statisticallysignificant when compared to the rate of entry into equivalent normalcells.

In some embodiments, the cupredoxin peptide is derived from, but is notlimited to, azurin, pseudoazurin, plastocyanin, rusticyanin, auracyanin,stellacyanin, cucumber basic protein or Laz. In particularly specificembodiments, the azurin is derived from Pseudomonas aeruginosa,Alcaligenes faecalis, Achromobacter xylosoxidans ssp. denitrificans I,Bordetella bronchiseptica, Methylomonas sp., Neisseria meningitidis,Neisseria gonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis,Xylella fastidiosa, Ulva pertussis or Vibrio parahaemolyticus. In a veryspecific embodiment, the azurin is derived from Pseudomonas aeruginosa.In other specific embodiments, the cupredoxin peptide comprises an aminoacid sequence that is SEQ ID NO: 1-25.

In some embodiments, the cupredoxin peptide has some of thepharmacologic activities of the P. aeruginosa azurin, and specificallyp28. In a specific embodiment, the cupredoxin peptide may inhibit thedevelopment of tumors, decrease the growth of tumors or kill tumor cellsin mammalian cells, tissues or animals. In some embodiments, themammalian tumor is composed of, but is not limited to, melanoma, breast,pancreas, glioblastoma, astrocytoma, lung, colorectal, neck and head,bladder, prostate, skin and cervical cancer cells. Inhibition of thedevelopment of tumors is any decrease, or lessening of the rate ofincrease, of the development of tumors that is statistically significantas compared to control treatments.

Cells of the Invention

The invention provides cells which express proteins of choice whencontacted by cancer cells. This aspect of the invention uses the azuringene from P. aeruginosa, which has been disclosed herein to be inducibleby contact with cancer cells. In one embodiment, the cell of theinvention is from P. aeruginosa, and harbors in its genome a copy of theP. aeruginosa azurin gene in which the coding sequence for azurin hasbeen replaced by the coding sequence for a target protein, and whichexpresses the target protein upon contact with cancer cells. In anotherembodiment, the cell may be a species other than P. aeruginosa, whichharbors in its genome a copy of the P. aeruginosa azurin gene, and whichexpresses P. aeruginosa azurin when contacted with cancer cells. Anexample of this embodiment is provided in Example 3. In anotherembodiment, the cell may be a species other than P. aeruginosa whichharbors in its genome a copy of the P. aeruginosa azurin gene in whichthe coding sequence for azurin has been replaced by the coding sequenceof a target protein, and which expresses the target protein upon contactwith cancer cells.

Target proteins of interest include therapeutic proteins, cytotoxicproteins and diagnostic proteins, and any other protein for whichproduction near cancer cells would be advantageous. Examples oftherapeutic proteins of interest include the cupredoxin peptidesprovided herein. Methods to make such cells are well known to those ofordinary skill in the art, and instructional manuals are available,including Sambrook et al., Molecular Cloning: A Laboratory Manual (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, Mass. (2001)).Nonetheless, the following non-limiting cell preparation and translationtreatment methods are provided.

Exemplary Host Cell Preparation Methodology.

Host cells to be transformed may be grown in any growth conducivemedium. Examples of such media include but are not limited to LuriaBroth, Luria Broth supplemented with 20 mM MgCl.sub.2, 0.001% thiamine,and 0.2% glucose; SOB medium containing 0.001% PPG (recipe given below);and 15/10 medium (recipe given below). Other suitable media will bereadily recognized by one of skill in the art.

The incubation temperatures for growing the cells may vary from about10° to about 42° C. In one embodiment, the temperature ranges from about12° to about 37° C. In another embodiment, the temperature ranges fromabout 15° C. to about 32° C., and in another embodiment from about 20°to about 25° C. In one particular embodiment, the cells may be grown atabout 23° C.

As one of ordinary skill in the art will understand, growth conditionsand culture age can affect both the viability and the transformationefficiency of cells following cryopreservation. Cells grown in shakeflask culture are generally more resistant to the stress offreeze-drying than are static broth cultures. Furthermore, the age of aculture also affects the ability of the culture to survivefreeze-drying. Generally, cells harvested in late log or earlystationary growth exhibit the greatest resistance to freeze-drying ifsuch freeze-drying is contemplated.

Thus, when freeze-drying is contemplated, the cells are preferably grownin shake flasks, although other means of growth may be used includingfermentators. Shake flasks used can be of any size and any type. In oneembodiment, baffled 2.8 liter shake flasks may be used for thisprocessing. Incubation times will vary according to the conditions used(temperature, medium, aeration, etc.) and the cell type. Aeration inflasks will also vary according to the rotation per minute (rpm) used,with higher rpms resulting in higher aeration. In certain embodiments,flasks are typically shaken at 100-500 rpms, at 200-400 rpms, and at200-300 rpms, although one of ordinary skill in the art may determineother preferred ranges. Cells are typically grown for a time and underconditions sufficient to reach an optical density (OD) at 550 nm betweenabout 0.1 to about 2.0. In one embodiment, the OD ranges from about 0.1to about 1.0. In another embodiment, the OD ranges from about 0.3 toabout 0.8. In another embodiment, the OD ranges from about from 0.5 toabout 0.8. In another embodiment, the OD ranges from about 0.5 to about0.7. In another embodiment, the OD ranges from about 0.6 to about 0.8.And in yet another embodiment, the OD ranges from about 0.66 to about0.75.

After the cells have reached the desired OD, the cells may be collectedfor further processing. If the desired OD is not reached or is exceeded,the cells can again be reinoculated and the growth process repeateduntil a culture of sufficient optical density is obtained.

After the cells are collected (by, without limitation, centrifugation,filtration, etc.), they may optionally be chilled (e.g., 0° to 4° C. for5 minutes to 2 hours). Collection of the cells may be accomplished bycentrifuging the cells to obtain a cell pellet. Collection may also beaccomplished by concentrating the cells and then centrifuging theconcentrated cultures to obtain a cell pellet. Methods of concentratingthe cells include, but are not limited to, dewatering the culture,filtering, or subjecting the culture to size exclusion chromatography,e.g. using by Centricon™ columns (Amicon Corp., Lexington, Mass.).

After the cells are collected, the cells may be resuspended in acompetence buffer. A competence buffer is any solution that enablescells to take up and establish exogenous DNA. Non-limiting examples ofcompetence buffers include 50 mM CaCl₂, 10 mM Tris/HCl (Maniatis, T. etal., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold SpringHarbor, N.Y. (1989)); 0.1 M MOPS (pH 6.5), 50 mM CaCl₂, 10 mM RbC1₂, 23%V/V DMSO; CCMB80 buffer (10 mM potassium acetate pH 7.0, 80 mMCaCl₂.H₂O, 20 mM MnCl₂4H₂O, 10 mM MgCl₂6H₂O, 10% glycerol adjusted to pH6.4 with 0.1 N HCl); TFB buffer (Liu et al., Biotechniques 8(1):23-25(1990)); and n1776 buffer (Maniatis, T. et al., Molecular Cloning: ALaboratory Manual (2nd ed., Cold Spring Harbor, N.Y. (1989)). Othersuitable buffers are disclosed in Tang et al., Nucl. Acids Res.22(14):2857-2858 (1994); Chung et al., Proc. Natl. Acad Sci. U.S.A.86:2172-2175 (1989); M. Dagert and S. D. Ehrlich, Gene 6:23-28 (1974);Kushner, S. R., In: Genetic Engineering (H. W. Boyer and S. Nicosia,eds.) pp. 17-23, Elsevier/North Holland, Amsterdam (1978); Mandel andHiga, J. Mol. Biol. 53:159-162 (1970); U.S. Pat. No. 4,981,797 byJessee; and Hanahan, D., J. Mol. Biol. 166:557-580 (1983) which areincorporated by reference herein.

The cells suspended in the competence buffer are incubated for asufficient time and at a temperature sufficient to make the cellscompetent to DNA uptake. In one embodiment, the cells are incubated atlow temperature (0 to 4° C.) for about 0 to about 3 hours. In anotherembodiment, the cells are incubated at low temperature (0 to 4° C.) forabout 5 minutes to about 1 hour. In another embodiment, the cells areincubated at low temperature (0 to 4° C.) for about 5 minutes to about30 minutes.

After the cells have been made competent, a cryoprotectant may be addeddirectly to the cell suspension. In one embodiment, the cells can becollected and then resuspended in a cryoprotectant. The concentration ofthe cryoprotectant can vary depending on the cell type, buffers used,the type of cryoprotectant and other factors. Optimal conditions can bedetermined by one of ordinary skill in the art without undueexperimentation. When used, cryoprotectants provide protection of thecells during the freezing process by depressing the freezing point,minimizing the effect of solution changes external to the cell,penetrating the cell to protect against solute concentration effects,and/or shifting the optimum cooling rate to lower values (F. P. Simione,Journal of Parenteral Science & Technology, 46(6):226-232 (1992)). Ofcourse, the cryoprotectant must not be toxic to the cells.

Any cryoprotectant and combination thereof may be and as will beapparent, the type and amount used may vary depending on the cell typeand conditions used. Cryoprotectants that can be used in the presentinvention include, but are not limited to, carbohydrates andcarbohydrate derivatives such as trehalose, sucrose, lactose, maltose,mannitol, galactose, ribose, fructose, xylose, mannose, dextrose,glucose, and sorbitol, and polymers such as polyethyleneamine,polyvinylpyrrolidone (PVP), ficoll, etc. Other cryoprotectants which canbe used in accordance with the invention, such as acacia gum, albumin,gelatin, and sugar alcohols, will be readily recognized by one skilledin the art.

After the cells have been mixed with the cryoprotectant, the cellsuspension may be aliquoted into containers to be used forlyophilization and storage, such as chilled cryovials, e.g., NUNC tubes(Gibco BRL, Gaithersburg, Md., Cat. No. 366656), or glass vials(Wheaton, Millville, N.J.). Prior to lyophilization, in one embodimentthe cells may be frozen at about −20° C. to about −180° C. In anotherembodiment they may be frozen at about −80° C. to about −180° C. In aparticular embodiment they may be frozen at about −80° C.

Methods of freezing a sample to a temperature from about −80° to about−180° C. are well known in the art. These include overnight storage(about 16 hours) of the vials which contain the cells in a −80° C.freezer, or immersion of the vials which contain the cells in dry ice,or in a low temperature bath, such as dry ice ethanol, or in a bathcontaining liquid nitrogen. Other such systems are disclosed in TheChemist's Companion: A Handbook of Practical Data, Techniques, andReferences, Gordon, A. J., et al., eds., John Wiley and Sons, NY (1972)which is incorporated by reference herein.

If desired, the cells can then be lyophilized by techniques which arewell known in the art. Lyophilization is a process by which ice and/ormoisture is removed from frozen cells by sublimation under vacuum atlow, subzero temperatures (e.g., −40° to −50° C.). Any residual moistureassociated with the “dried” preparation is then removed by graduallyraising the temperature, resulting in evaporation. Thus, lyophilizationcomprises subjecting frozen cells to a vacuum under conditionssufficient to substantially remove moisture and/or ice from said cells(also referred to herein as substantially dried cells). Thesubstantially dried cells may then be stored at various temperatures(room temperature to about −180° C., and in certain embodiments, fromabout 4° C. to about −80° C., from about −20° C. to about −80° C., andin one particular embodiment at about −20° C.).

One non-limiting example of a cell lyophilization process comprises thesteps of (a) loading a container containing frozen cells into alyophilizer, the lyophilizer having a temperature of about −40° to about−50° C.; (b) subjecting the cells to a vacuum; and (c) substantiallydrying the cells. In one embodiment the vacuum is less than about 100μm, and the cells are dried by: (i) holding the temperature of thechamber at about −45° C. for about 2 hours; and (ii) increasing thetemperature of the chamber from about −45° C. to about 10° C. at therate of about 0.1° to about 1.0° C./hr (in one embodiment, about 0.5° toabout 0.8° C./hr, and in a particular embodiment about 0.6° to about0.8° C./hr). The cell container may then be sealed and stored forextended time at various temperatures.

The viable host cell count of competent cells produced by the describedmethod should remain at greater than about 1×10⁷ to about 1×10⁹ cells/mlwhen stored at −20° C. for any time period from about 0 days to about450 days. The cells likely retain a transformation efficiency of atleast about 1×10⁵ and preferably at least about 1×10⁹ transformants permicrogram of DNA (T/μg). Suitable storage temperatures vary from aboutroom temperature to about −180° C. In one embodiment the storagetemperature ranges from about 4° C. to about −80° C. In anotherembodiment the storage temperature ranges from about −20° C. to about−80° C. In another particular embodiment the storage temperature rangesfrom about −20° C. The storage period or time may range from about 0days to about 45 days, from about 0 days to about 90 days, from about 0days to about 150 days, from about 240 days to about 365 days, or fromabout 365 days to about 450 days, although longer storage times may beused at temperatures of about −20° C. and below. Competent host cellsproduced by this method may be stored at −20° C. for at least one yearwhile retaining substantially their transformation efficiency.

Exemplary Host Cell Transformation Methodology. In order to transformhost cells, and as will be understood by those of ordinary skill in theart, generally the cells can be mixed with a DNA molecule of interestand incubated under conditions sufficient to transform the cells withsaid DNA molecule. Any DNA molecules (e.g. vectors, plasmids, phagemids,expression vectors, etc.) may be used. Preferably, the cells are mixedwith the DNA molecule in the presence of a competence buffer. Thecompetence buffer may be added to competent host cells prior to addingthe DNA molecule or the DNA molecule and competence buffer may be addedsimultaneously to the competent host cells. Although mixing thecompetent host cells with the DNA molecule and a competence buffer ispreferred, any solution may be used to rehydrate and mix the cells withthe DNA molecule of interest. Such solutions include water, saline, orany suitable buffer.

The following more detailed non-limiting example is also provided. About25-200 μl cells can be thawed and kept on ice. The cells should not beplaced in glass tubes as glass will adsorb the DNA of interest. DNA canbe gently pipetted into the cell mixture, keeping the volume of DNA toless than about 5% of the cell volume. The cells can be incubated withthe DNA of interest for about 5 to about 30 minutes. Next, the cells canbe incubated for about 30 seconds at about 42° C. and then incubated forabout 2 minutes on ice. At this stage, about 4 volumes of SOC Medium canbe added but this addition should not be critical to the success of thetransformation. The cells can then be incubated for about 30 minutes toabout 1 hour on a shaker at 37° C. Following this incubation, about100-300 μl of the cell/DNA mixture can be spread onto a plate made withan appropriate antibiotic, if desired.

Once the cells have been transformed with the DNA molecule of interest,the transformed cells may be grown in a growth conducive medium.Typically, such a growth conducive medium will also contain anantibiotic to assist in selection of the transformed cells. That is, theDNA molecule to be transformed may contain a selective marker (e.g. anantibiotic resistance gene), allowing selection of the transformed cellswhen the corresponding antibiotic is used in the medium. These aspectsof the process are not, however, required.

When transformed host cells (and condensed DNA) are used as a treatmentaccording to the present invention, particularly useful administrationmethods may include, without limitation, local delivery through acatheter and drug pump system, delivery by direct local injection orthrough the use of polymers. In one embodiment, a “controlledadministration system” including a direct and local administrationsystem can be used. A controlled administration system can be a depot ora pump system, such as, without limitation, an osmotic pump or aninfusion pump. An infusion pump can be implantable and can be, withoutlimitation, a programmable pump, a fixed rate pump, and the like. Acatheter can be operably connected to the pump and configured to delivercells of the present invention to a target tissue region of a subject.Other delivery methods described throughout the remainder of thespecification may also be appropriate.

Methods of Use

The invention provides methods to treat patients, specifically patientssuffering from a condition, specifically cancer, and to prevent cancerin patients, comprising co-administering to said patients CpG rich DNAfrom P. aeruginosa and at least one cupredoxin peptide. Such treatmentcan be carried out by administering at least one of the isolated CpGrich polynucleotides of the invention. Cancers that may be prevented ortreated by treatment with the compositions of the invention include, butare not limited to, melanoma, breast, pancreas, glioblastoma,astrocytoma, lung, colorectal, neck and head, bladder, prostate, skin,and cervical cancer. In other embodiments, the patients are sufferingfrom AIDS, malaria, inappropriate angiogenesis, or are at a higher atrisk to develop cancer than the general population. In some embodiments,the patient may be human. In other embodiments, the patient is nothuman.

The compositions comprising at least one CpG rich DNA from P. aeruginosaand at least one cupredoxin peptide can be administered to the patientby many routes and in many regimens that will be well known to those inthe art. In specific embodiments, the CpG rich DNA from P. aeruginosaand cupredoxin peptide are administered intravenously, intramuscularly,subcutaneously, topically, orally, or by inhalation. The compositionsmay be administered to the patient by any means that delivers thepolynucleotides and polypeptides to the patient. In some embodiments,the patient is suffering from cancer and the compositions areadministered in a manner that delivers the polynucleotides andpolypeptides to the site of the tumor. In specific embodiments, the CpGrich DNA from P. aeruginosa and cupredoxin peptide are administeredintravenously.

In some embodiments, the methods of the invention comprise administeringto a patient at least one composition comprising one unit dose of acupredoxin peptide and one unit dose of a CpG rich DNA from P.aeruginosa.

In other embodiments, the methods may comprise co-administering to apatient one unit dose of at least one composition comprising acupredoxin peptide and one unit dose of at least one compositioncomprising a CpG rich DNA from P. aeruginosa in either order,administered at about the same time, or within about a given timefollowing the administration of the other, for example, about one minuteto about 60 minutes following the administration of the other drug, orabout 1 hour to about 12 hours following the administration of the otherdrug.

In other embodiments, the methods may comprise co-administering to apatient one unit dose of at least one composition comprising acupredoxin peptide, one unit dose of at least one composition comprisinga CpG rich DNA from P. aeruginosa, and one unit dose of a compositioncomprising another prophylactic or therapeutic drug, in either order,administered at about the same time, or within about a given timefollowing the administration of the others, for example, about oneminute to about 60 minutes following the administration of the otherdrug, or about 1 hour to about 12 hours following the administration ofthe other drug. The additional therapeutic drug may be one or more ofmany that are commonly used to treat the condition suffered by thepatient or the side effects of the treatment. Such therapeutic drugsinclude, but are not limited to, those used to treat cancer, AIDS,malaria, inappropriate angiogenesis, inflammatory bowel disease, viraldiseases, cardiovascular disease, peripheral vascular diseases, centralnervous system disorders, degeneration of the central nervous system andAlzheimer's disease and to prevent cancer. Examples of such therapeuticdrugs are provided in U.S. Pat. No. 7,084,105, U.S. Patent ApplicationPublication No. 20060040269, published Feb. 23, 2006, now U.S. Pat. No.7,491,394; U.S. Patent Application Publication No. 20060149037,published Jul. 6, 2006, now U.S. Pat. No. 7,691,383; U.S. PatentApplication Publication No. 20080213185, published Sep. 4, 2008, nowU.S. Pat. No. 7,807,183; U.S. Patent Application Publication No.20080089878, published Apr. 17, 2008, now U.S. Pat. No. 7,381,701; U.S.Patent Application Publication No. 20080103087, published May 1, 2008,now U.S. Pat. No. 7,556,810; U.S. Patent Application Publication No.20060251669, now U.S. Pat. No. 7,338,766; U.S. Patent ApplicationPublication No. 20060251639, published Nov. 9, 2006, now U.S. Pat. No.7,301,010; U.S. Patent Application Publication No. 20100267608,published Oct. 21, 2010; and U.S. Patent Application Publication No.20080139471, published Jun. 12, 2008, now U.S. Pat. No. 7,618,939, eachof which in incorporated herein by reference. Further therapeutic drugsof interest may be found in the Thomson Physician's Desk Reference(Thomson Healthcare, Stamford Conn., 2006), and other references thatare well known to those of ordinary skill in the art.

Drugs for treating cancer include but are not limited to mechlorethaminehydrochloride, cyclophosphamide, chlorambucil, melphalan, ifosfamide,busulfan, carmustin, lomustine, semustine, streptozocin, thiotepa,dacarbazine, methotrexate, thioguanine, mercaptopurine, fludarabine,pentastatin, cladribin, cytarabine, fluorouracil, doxorubicinhydrochloride, daunorubicin, idarubicin, bleomycin sulfate, mitomycin C,actinomycin D, safracins, saframycins, quinocarcins, discodermolides,vincristine, vinblastine, vinorelbine tartrate, etoposide, etoposidephosphate, teniposide, paclitaxel, tamoxifen, estramustine, estramustinephosphate sodium, flutamide, buserelin, leuprolide, pteridines,diyneses, levamisole, aflacon, interferon, interleukins, aldesleukin,filgrastim, sargramostim, rituximab, BCG, tretinoin, irinotecanhydrochloride, betamethosone, gemcitabine hydrochloride, altretamine,and topoteca and any analogs or derivatives thereof.

Preferred members of these classes include, but are not limited to,paclitaxel, cisplatin, carboplatin, doxorubicin, carminomycin,daunorubicin, aminopterin, methotrexate, methopterin, mitomycin C,ecteinascidin 743, or pofiromycin, 5-fluorouracil, 6-mercaptopurine,gemcitabine, cytosine arabinoside, podophyllotoxin or podophyllotoxinderivatives such as etoposide, etoposide phosphate or teniposide,melphalan, vinblastine, vincristine, leurosidine, vindesine andleurosine.

Drugs for treating cancer include the following: epothilone derivativesas found in German Patent No. 4138042.8; WO 97/19086, WO 98/22461, WO98/25929, WO 98/38192, WO 99/01124, WO 99/02224, WO 99/02514, WO99/03848, WO 99/07692, WO 99/27890, WO 99/28324, WO 99/43653, WO99/54330, WO 99/54318, WO 99/54319, WO 99/65913, WO 99/67252, WO99/67253 and WO 00/00485; cyclin dependent kinase inhibitors as found inWO 99/24416 (see also U.S. Pat. No. 6,040,321); and prenyl-proteintransferase inhibitors as found in WO 97/30992 and WO 98/54966; andagents such as those described generically and specifically in U.S. Pat.No. 6,011,029 (the compounds of which U.S. patent can be employedtogether with any NHR modulators (including, but not limited to, thoseof present invention) such as AR modulators, ER modulators, with LHRHmodulators, or with surgical castration, especially in the treatment ofcancer).

Drugs for treating HIV infection include, but are not limited to,reverse transcriptase inhibitors: AZT (zidovudine), ddC (zalcitabine,dideoxyinosine), d4T (stavudine), and 3TC (lamivudine), nonnucleosidereverse transcriptase inhibitors (NNRTIS): delavirdine and nevirapine,protease inhibitors: ritonavir, a lopinavir and ritonavir combination,saquinavir, indinavir sulphate, amprenavir, and nelfinavir. Presently, acombination of several drugs called highly active antiretroviral therapy(HAART) is used to treat people with HIV.

Drugs for treating malaria include, but are not limited to, proguanil,chlorproguanil, trimethoprim, chloroquine, mefloquine, lumefantrine,atovaquone, pyrimethamine-sulfadoxine, pyrimethamine-dapsone,halofantrine, quinine, quinidine, amodiaquine, amopyroquine,sulphonamides, artemisinin, arteflene, artemether, artesunate,primaquine, pyronaridine, proguanil, chloroquine, mefloquine,pyrimethamine-sulfadoxine, pyrimethamine-dapsone, halofantrine, quinine,proguanil, chloroquine, mefloquine,1,16-hexadecamethylenebis(N-methylpyrrolidinium)dibromide, andcombinations thereof.

Drugs for treating with inflammatory bowel disease, include, but are notlimited to, aminosalicylates, such as, sulfasalazine, olsalazine,mesalamine, and balsalazide; corticosteroids, such as, prednisone,methylprednisolone, hydrocortisone, Budesonide; immunomodulators, suchas, azathioprine, 6-mercaptopurine (6-MP) and cyclosporine A;antibiotics, such as, metronidazole and ciprofloxacin; biologictherapies, such as, infliximab; and miscellaneous therapies, such as,tacrolimus (FK506) and mycophenolate mofetil.

Drugs for treating HIV infection include, but are not limited to,reverse transcriptase inhibitors: AZT (zidovudine), ddC (zalcitabine,dideoxyinosine), d4T (stavudine), and 3TC (lamivudine), nonnucleosidereverse transcriptase inhibitors (NNRTIS): delavirdine and nevirapine,protease inhibitors: ritonavir, a lopinavir and ritonavir combination,saquinavir, indinavir sulphate, amprenavir, and nelfinavir.

Drugs for treating viral diseases include, but are not limited to,acyclovir, varicella zoster immune globulin, peginterferon, ribavirin,acyclovir, valacyclovir, famciclovir, amantadine, rimantadine,zanamivir, oseltamivir, and alpha interferon.

Drugs for treating cardiovascular disorders include, but are not limitedto, anticoagulants, antiplatelet agents, thrombolytic agents, adrenergicblockers, adrenergic stimulants, alpha/beta adrenergic blockers,angiotensin converting enzyme (ACE) inhibitors, angiotensin convertingenzyme (ACE) inhibitors with calcium channel blockers, angiotensinconverting enzyme (ACE) inhibitors with diuretics, angiotensin IIreceptor antagonists, calcium channel blockers, diuretics (includingcarbonic anhydrase inhibitors, loop diuretics, potassium-sparingdiuretics, thiazides and related diuretics, vasodilators, vasopressors,etc.

Drugs for treating peripheral vascular disease include, but are notlimited to, pentoxifylline, an oral methylxanthine derivative, andcilostazol, a phosphodiesterase III inhibitor;antiplatelet/antithrombotic therapy such as aspirin; anticoagulants suchas heparin and warfarin; cholesterol lowering drugs, such as, niacin,statins, fibrates, gemfibrozil; fenofibrate; bile acid sequestrants,micronized colestipol hydrochloride; colesevelam hydrochloride; calciumchannel blockers; vitamins and dietary supplements, such as, folate,B-6, B-12, L-arginine and omega-3 fatty acids; and HMG-COA Reductaseinhibitors, such as, Niacin/Lovastatin; lovastatin; fluvastatin sodium;atorvastatin; lovastatin; Pravastatin sodium; Buffered Aspirin andPravastatin Sodium; Simvastatin; Merck); nicotinic acid agents, such as,Niacin/Lovastatin (also listed as a HMG-COA Reductase inhibitor);niacin; and miscellaneous agents, such as, ezetimibe.

Drugs for treating central nervous system disorders include, but are notlimited to, psychotherapeutic agents, such as, various benzodiazepinepreparations and combinations, antianxiety agents, antidepressants(including monoamine oxidase inhibitors (MAOI), selective serotoninreuptake inhibitors (SSRIs), tricyclic antidepressants), antimanicagents, antipanic agents, antipsychotic agents, psychostimulants, andobsessive-compulsive disorder management agents; migraine preparations,such as beta adrenergic blocking agents, isometheptene and serotoninreceptor agonists, as well as miscellaneous migraine preparationsincluding Divalproex sodium and acetaminophen; sedatives and hypnotics;anticonvulsants; and pimozide. Drugs for treating Parkinson's diseaseinclude, but are not limited to, anticholinergic agents,catechol-o-methyltransferase inhibitors, dopamine agents and monoamineoxidase (MAO) inhibitors.

Drugs for treating Central Nervous System (CNS) degeneration disordersinclude the following: Drugs for treating Multiple Sclerosis include,but are not limited to, interferon beta-1a; BETASERON for SC injection(modified form of Interferon beta-1b; Berlex); Glatiramer Acetate;Methylprednisolone acetate; Mitoxantrone supplied as mitoxantronehydrochloride; prednisolone sodium phosphate oral solution. Drugs fortreating Huntington's Disease include, but are not limited to,tranquilizers such as clonazepam; antipsychotic drugs such ashaloperidol and clozapine; fluoxetine, sertraline, nortriptyline, andlithium.

Drugs for treating Alzheimer's disease include, but are not limited to,Donepezil Hydrochloride; rivastigmine (as the hydrogen tartrate salt);rivastigmine tartrate; galantamine hydrobromide; or galantaminehydrobromide.

Chemopreventive drugs of interest include, but are not limited to,tamoxifen, aromatase inhibitors such as letrozole and anastrozole,retinoids such as N-[4-hydroxyphenyl] retinamide (4-HPR, fenretinide),nonsteriodal antiinflammatory agents (NSAIDs) such as aspirin andsulindac, celecoxib (COX-2 inhibitor), defluoromethylornithing (DFMO),ursodeoxycholic acid, 3-hydroxy-3-methylglutaryl coenzyme A reductaseinhibitors, EKI-785 (EGFR inhibitor), bevacizumab (antibody toVEGF-receptor), cetuximab (antibody to EGFR), retinol such as vitamin A,beta-carotene, 13-cis retinoic acid, isotretinoin and retinyl palmitate,α-tocopherol, interferon, oncolytic adenovirus dl1520 (ONYX-015),gefitinib, etretinate, finasteride, indole-3-carbinol, resveratrol,chlorogenic acid, raloxifene, and oltipraz.

The methods of the invention further comprise methods of administeringthe cells of the invention. Such methods include methods for treatingpatients, specifically suffering from conditions, specifically cancer,and methods for diagnosing cancer in patients. In some embodiments,patients suffering from cancer may be treated by administering either acell expressing azurin when in contact with cancer cells, or expressinga therapeutic or cytotoxic target protein when in contact with cancercells. In other embodiments, the patients are suffering from cancer,such as, but not limited to, melanoma, breast, pancreas, glioblastoma,astrocytoma, lung, colorectal, neck and head, bladder, prostate, skin,and cervical cancer. In other embodiments, the method diagnoses cancerin a patient by administering a cell expressing a diagnostic proteinwhen contacted by cancer cells, and then detecting the diagnosticprotein, either in the blood stream or at its site of expression.

The cells of the invention may be administered to patients by any meanssuitable, including but not limited to, intravenous injection,intramuscular injection, subcutaneous injection, inhalation, topicaladministration, suppository, vitreous injection and oral. In a specificembodiment, the cells are administered intravenously.

Pharmaceutical Compositions Comprising CpG Rich DNA or Cells

Pharmaceutical compositions comprising CpG rich DNA from P. aeruginosaor the cells of the invention may be manufactured in any suitableconventional manner, e.g., by conventional mixing, dissolving,granulating, dragee-making, emulsifying, encapsulating, entrapping, orlyophilizing processes. The substantially pure or pharmaceutical gradeCpG rich DNA from P. aeruginosa or the cells of the invention can bereadily combined with a pharmaceutically acceptable carrier well-knownin the art. Such carriers enable the preparation to be formulated as atablet, pill, dragee, capsule, liquid, gel, syrup, slurry, suspension,and the like. Suitable carriers or excipients can also include, forexample, fillers and cellulose preparations. Other excipients caninclude, for example, flavoring agents, coloring agents, detackifiers,thickeners, and other acceptable additives, adjuvants, or binders. Insome embodiments, the pharmaceutical preparation is substantially freeof preservatives. In other embodiments, the pharmaceutical preparationmay contain at least one preservative. General methodology onpharmaceutical dosage forms is found in Ansel et al., PharmaceuticalDosage Forms and Drug Delivery Systems (Lippencott Williams & Wilkins,Baltimore Md. (1999)).

The composition comprising CpG rich DNA from P. aeruginosa or the cellsof the invention may be administered in a variety of ways, including byinjection (e.g., intradermal, subcutaneous, intramuscular,intraperitoneal and the like), by inhalation, by topical administration,by suppository, by using a transdermal patch or by mouth. Generalinformation on drug delivery systems can be found in Ansel et al., id.In some embodiments, the composition comprising CpG rich DNA from P.aeruginosa or the cells of the invention can be formulated and useddirectly as injectibles, for subcutaneous and intravenous injection,among others. The injectable formulation, in particular, canadvantageously be used to treat patients that are appropriate forchemopreventive therapy. The composition comprising a CpG rich DNA fromP. aeruginosa or the cells of the invention can also be taken orally,optionally after mixing with protective agents such as polypropyleneglycols or similar coating agents.

When administration is by injection, the CpG rich DNA from P. aeruginosaor the cells of the invention may be formulated in aqueous solutions,specifically in physiologically compatible buffers such as Hankssolution, Ringer's solution, or physiological saline buffer. Thesolution may contain formulatory agents such as suspending, stabilizingand/or dispersing agents. Alternatively, the CpG rich DNA from P.aeruginosa may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use. In someembodiments, the pharmaceutical composition does not comprise anadjuvant or any other substance added to enhance the immune responsestimulated by the polynucleotide. In some embodiments, thepharmaceutical composition comprises a substance that inhibits an immuneresponse to the polynucleotide.

When administration is by intravenous fluids, the intravenous fluids foruse administering the CpG rich DNA from P. aeruginosa or the cells ofthe invention may be composed of crystalloids or colloids. Crystalloidsas used herein are aqueous solutions of mineral salts or otherwater-soluble molecules. Colloids as used herein contain largerinsoluble molecules, such as gelatin. Intravenous fluids may be sterile.

Crystalloid fluids that may be used for intravenous administrationinclude but are not limited to, normal saline (a solution of sodiumchloride at 0.9% concentration), Ringer's lactate or Ringer's solution,and a solution of 5% dextrose in water sometimes called D5W, asdescribed in Table 1.

TABLE 1 Composition of Common Crystalloid Solutions Solution Other Name[Na⁺] [Cl⁻] [Glucose] D5W 5% Dextrose 0 0 252 ⅔ & ⅓ 3.3% Dextrose/ 51 51168 0.3% saline Half-normal 0.45% NaCl 77 77 0 saline Normal saline 0.9%NaCl 154 154 0 Ringer's Ringer's 130 109 0 lactate* solution *Ringer'slactate also has 28 mmol/L lactate, 4 mmol/L K⁺ and 3 mmol/L Ca²⁺.

When administration is by inhalation, the CpG rich DNA from P.aeruginosa or the cells of the invention may be delivered in the form ofan aerosol spray from pressurized packs or a nebulizer with the use of asuitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin, for use in an inhaler or insufflator may beformulated containing a powder mix of the polynucleotides and a suitablepowder base such as lactose or starch.

When administration is by topical administration, the CpG rich DNA fromP. aeruginosa or the cells of the invention may be formulated assolutions, gels, ointments, creams, jellies, suspensions, and the like,as are well known in the art. In some embodiments, administration is bymeans of a transdermal patch. When administration is by suppository(e.g., rectal or vaginal), CpG rich DNA from P. aeruginosa or the cellsof the invention compositions may also be formulated in compositionscontaining conventional suppository bases.

When administration is oral, the CpG rich DNA from P. aeruginosa or thecells of the invention can be readily formulated by combining the CpGrich DNA from P. aeruginosa or the cells of the invention withpharmaceutically acceptable carriers well known in the art. A solidcarrier, such as mannitol, lactose, magnesium stearate, and the like maybe employed; such carriers enable the CpG rich DNA from P. aeruginosa orthe cells of the invention to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, fororal ingestion by a subject to be treated. For oral solid formulationssuch as, for example, powders, capsules and tablets, suitable excipientsinclude fillers such as sugars, cellulose preparation, granulatingagents, and binding agents.

Other convenient carriers, as well-known in the art, also includemultivalent carriers, such as bacterial capsular polysaccharide, adextran or a genetically engineered vector. In addition,sustained-release formulations that include a CpG rich DNA from P.aeruginosa allow for the release of the CpG rich DNA over extendedperiods of time, such that without the sustained release formulation,the CpG rich DNA from P. aeruginosa would be cleared from a subject'ssystem, and/or degraded by, for example, proteases and simple hydrolysisbefore eliciting or enhancing a therapeutic effect.

In various embodiments, the pharmaceutical composition includes carriersand excipients (including but not limited to buffers, carbohydrates,mannitol, proteins, polypeptides or amino acids such as glycine,antioxidants, bacteriostats, chelating agents, suspending agents,thickening agents and/or preservatives), water, oils, saline solutions,aqueous dextrose and glycerol solutions, other pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions, such as buffering agents, tonicity adjusting agents, wettingagents and the like. It will be recognized that, while any suitablecarrier known to those of ordinary skill in the art may be employed toadminister the compositions of this invention, the type of carrier willvary depending on the mode of administration. Compounds may also beencapsulated within liposomes using well-known technology. Biodegradablemicrospheres may also be employed as carriers for the pharmaceuticalcompositions of this invention. Suitable biodegradable microspheres aredisclosed, for example, in U.S. Pat. Nos. 4,897,268; 5,075,109;5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344 and 5,942,252.

The pharmaceutical compositions comprising CpG rich DNA may besterilized by conventional, well-known sterilization techniques, or maybe sterile filtered. The resulting aqueous solutions may be packaged foruse as is, or lyophilized, the lyophilized preparation being combinedwith a sterile solution prior to administration.

Administration of CpG Rich DNA or Cells

The CpG rich DNA from P. aeruginosa or cells of the invention can beadministered formulated as pharmaceutical compositions and administeredby any suitable route, for example, by oral, buccal, inhalation,sublingual, rectal, vaginal, transurethral, nasal, topical,percutaneous, i.e., transdermal or parenteral (including intravenous,intramuscular, subcutaneous and intracoronary) or vitreousadministration. The pharmaceutical formulations thereof can beadministered in any amount effective to achieve its intended purpose.More specifically, the composition is administered in a prophylacticallyor therapeutically effective amount. In specific embodiments, theprophylactically or therapeutically effective amount is generally fromabout 0.01-20 mg/day/kg of body weight.

The compounds comprising CpG rich DNA from P. aeruginosa or cells of theinvention are useful for the treatment of conditions, and specificallycancer, and preventing cancer, alone or in combination with other activeagents. The appropriate dosage will, of course, vary depending upon, forexample, the compound of CpG rich DNA from P. aeruginosa or the cells ofthe invention employed, the host, the mode of administration and thenature and severity of the condition. However, in general, satisfactoryresults in humans are indicated to be obtained at daily dosages fromabout 0.01-20 mg/kg of body weight. An indicated daily dosage in humansis in the range from about 0.7 mg to about 1400 mg of a compound of CpGrich DNA from P. aeruginosa or the cells of the invention convenientlyadministered, for example, in daily doses, weekly doses, monthly doses,and/or continuous dosing. Daily doses can be in discrete dosages from 1to 12 times per day. Alternatively, doses can be administered everyother day, every third day, every fourth day, every fifth day, everysixth day, every week, and similarly in day increments up to 31 days orover. Alternatively, dosing can be continuous using patches, i.v.administration and the like.

The exact formulation, route of administration, and dosage is determinedby the attending physician in view of the patient's condition. Dosageamount and interval can be adjusted individually to provide plasmalevels of the CpG rich DNA from P. aeruginosa and cupredoxin peptidewhich are sufficient to maintain prophylactic or therapeutic effect.Generally, the desired CpG rich DNA from P. aeruginosa is administeredin an admixture with a pharmaceutical carrier selected with regard tothe intended route of administration and standard pharmaceuticalpractice. In the case of the cells, of the invention, dosage andinterval can be adjusted individually to provide levels of the targetprotein or azurin at the site of the cancer cells which are sufficientto maintain a prophylactic or therapeutic effect or diagnostic presence.

Kits Comprising CpG Rich DNA or Cells

In one aspect, the invention provides regimens or kits comprising one ormore of the following in a package or container: (1) a pharmaceuticalcomposition comprising CpG rich DNA from P. aeruginosa; (2) apharmaceutical composition comprising at least one cupredoxin peptide;(3) an additional prophylactic or therapeutic drug; and (4) apparatus toadminister the biologically active composition to the patient, such as asyringe, nebulizer etc.

In another aspect, the invention provides regimens or kits comprisingone or more of the following in a package or container: (1) a cell ofthe invention; and (2) an apparatus to administer the cells to thepatient.

When a kit is supplied, the different components of the composition maybe packaged in separate containers, if appropriate, and admixedimmediately before use. Such packaging of the components separately maypermit long-term storage without losing the active components'functions.

The reagents included in the kits can be supplied in containers of anysort such that the life of the different components are preserved andare not adsorbed or altered by the materials of the container. Forexample, sealed glass ampules may contain lyophilized CpG rich DNA, orbuffers that have been packaged under a neutral, non-reacting gas, suchas nitrogen. Ampules may consist of any suitable material, such asglass, organic polymers, such as polycarbonate, polystyrene, etc.,ceramic, metal or any other material typically employed to hold similarreagents. Other examples of suitable containers include simple bottlesthat may be fabricated from similar substances as ampules, andenvelopes, that may comprise foil-lined interiors, such as aluminum oran alloy. Other containers include test tubes, vials, flasks, bottles,syringes, or the like. Containers may have a sterile access port, suchas a bottle having a stopper that can be pierced by a hypodermicinjection needle. Other containers may have two compartments that areseparated by a readily removable membrane that upon removal permits thecomponents to be mixed. Removable membranes may be glass, plastic,rubber, etc.

Kits may also be supplied with instructional materials. Instructions maybe printed on paper or other substrate, and/or may be supplied as anelectronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, Zipdisc, videotape, audiotape, flash memory device etc. Detailedinstructions may not be physically associated with the kit; instead, auser may be directed to an internet web site specified by themanufacturer or distributor of the kit, or supplied as electronic mail.

A more complete understanding of the present invention can be obtainedby reference to the following specific Examples. The Examples aredescribed solely for purposes of illustration and are not intended tolimit the scope of the invention. Changes in form and substitution ofequivalents are contemplated as circumstances may suggest or renderexpedient. Although specific terms have been employed herein, such termsare intended in a descriptive sense and not for purposes of limitations.Modifications and variations of the invention as hereinbefore set forthcan be made without departing from the spirit and scope thereof.

EXAMPLES Example 1 Pseudomonas aeruginosa Released Azurin into theMedium in the Presence of MCF-7 Cells

Azurin from Pseudomonas aeruginosa is a periplasmic protein but is knownto be secreted in the growth medium at the late stage of growth.Zaborina et al., Microbiology 146:2521-2530 (2000). Its extracellularrelease is dependent on quorum sensing at high cell density andmodulated by GacA or the two small RNA products RsmY and RsmZ. Kay etal., J. Bacteriol. 188:6026-6033 (2006). Experiments were performed todetermine if P. aeruginosa released azurin into the extracellular mediumwhen contacted with cancer cells.

A mucoid isolate strain 8821 of P. aeruginosa from a cystic fibrosispatient and its spontaneous nonmucoid (nonalginate secreting) derivative8822 were used as described previously by Darzins et al. (J. Bacteriol.161:249-257 (1985)). When grown in a glucose-minimal medium supplementedwith histidine, strain 8822 secreted azurin in the growth medium at themid exponential growth phase after about 4 hours of growth. No azurinwas seen in the growth medium during the lag or very early log phase ofabout 2 hours of growth. The mucoid strain 8821 was deficient in azurinsecretion even at late log or early stationary phase. It is known thatmucoid strains are deficient in extracellular protein secretion becauseof copious secretion of the exopolysaccharide alginate. Ohman andChakrabarty, Infect. Immun. 37:662-669 (1982); Woods et al., J. Infect.Dis. 163:143-149 (1991).

1.6×10⁴ to 4×10⁵ cells of the human breast cancer cell line MCF-7 wereadded to the 8821 and 8822 cells (at an optical density at 660 nm ofabout 0.3), grown for several hours and measured the level of azurin byWestern blotting using anti-azurin antibodies in the extracellulargrowth medium after centrifugation of the cells and filtering the growthmedium through a 0.22 μm membrane filter. Punj et al., Oncogene23:2367-2378 (2004). A control without the MCF-7 cells but with anequivalent amount of the growth medium used.

The addition of MCF-7 cells to either mucoid strain 8821 or thenonmucoid strain 8822 had very little effect on the growth rates of thePseudomonas cells (FIG. 1A, a). Most interestingly, however, theexposure to the MCF-7 cells elicited significant amounts of azurinsecretion to the extracellular growth medium within 20 min (FIG. 1A, b),as determined by Western blotting (FIG. 1A, c), but only from strain8822. No secretion of azurin was seen in absence of the MCF-7 cells(FIG. 1A, b and c) up to an hour or for several hours in the mucoidstrain 8821 which is known to be deficient in extracellular proteinsecretion. Quantization of azurin secretion by Western blotting was doneusing a standard curve (FIG. 1B). Similar results were obtained withanother human cancer cell line melanoma Mel-2 (data not shown). In caseof both MCF-7 and Mel-2, the secretion of azurin was observed within 20to 30 min of exposure to the cancer cells, but no secretion in absenceof the cancer cells, strongly indicated that strain 8822 is capable ofsensing the presence of human cancer cells and starts to secrete azurinin response to their presence.

Similar secretion, albeit delayed to 40 min or so, was seen in presenceof MCF-10A cells, which are immortalized normal breast epithelial cells.The extent of azurin secretion was also dependent upon theconcentrations of the cancer cells: a much higher level of azurin wasseen in growth medium when 1×10⁸/ml 8822 cells were exposed to 2×10⁶/mlcancer cells (1.4 nM azurin) than when exposed to 1.6×10⁴/ml MCF-7 cells(<0.1 nM azurin) or 8×10⁴ cells/ml (0.7 nM azurin), showing adose-dependence of azurin secretion on exposure to MCF-7 cells.

To ensure that the mucoid strain 8821 was not deficient in azurinproduction, the intracellular levels of azurin were also measured inboth strains 8821 and 8822 (FIG. 1C) by making cellular lysates, andseparating the azurin by SDS-PAGE using a fixed amount (50 μg) of thelysate protein. Both strains 8821 and 8822 demonstrated the presence ofintracellular (periplasmic) azurin, although secretion was observed onlyfrom the nonmucoid cells. Quantization of azurin from the standard curve(FIG. 1B) in the intracellular and extracellular fractions of strain8822 grown in absence (control) or in presence of MCF-7 cellsdemonstrated that about 6 to 8% of intracellular azurin was released tothe external medium during the 60 min incubation period.

Example 2 MCF-7 Cells Induce Azurin Release from P. aeruginosa withoutCell Lysis

To determine if addition of the cancer cells may induce lysis in P.aeruginosa, thereby releasing azurin and other proteins in the growthmedium, into strain 8822 was introduced the broad host range plasmidpQF47 which harbors a functional lacZ gene encoding β-galactosidase. Therelease of azurin and β-galactosidase (LacZ) in the growth medium wasthen assessed in absence and in presence of MCF-7 cells during a 60 minperiod.

There was no measurable release into the extracellular growth medium ofLacZ either in absence (control) or in presence of MCF-7 cells duringthe 60 min incubation period (FIG. 2A). Examination of the cellularlysates demonstrated the presence of intracellular LacZ either inabsence or in presence of cancer cells (FIG. 2A), indicating very littlecellular lysis and therefore very little release of the intracellularLacZ into the external growth medium.

An examination of the presence of azurin in the growth medium, however,clearly indicated that while azurin was secreted and therefore detectedin presence of MCF-7 cells, very little azurin was detected in itsabsence (FIG. 2B), clearly demonstrating the role of MCF-7 cells ininducing azurin secretion, but not LacZ release, from the cells. Anestimation of intracellular LacZ in both strain 8822 alone and in strain8822 harboring the pQF47 plasmid (8822/pQF47) demonstrated, as expected,that while the P. aeruginosa strain 8822 lacked a functional lacZ geneand therefore produced no LacZ protein, 8822/pQF47 produced significantquantities of intracellular LacZ (FIG. 2C). That the presence of azurinin the external medium is not due to lysis is also confirmed by the factthat the mucoid strain 8821, normally deficient in protein secretion, isalso deficient in azurin secretion (FIG. 1A): lysis of the cells wouldhave released azurin in its growth medium.

Concentrated filtered growth medium of MCF-7 cells was used to determineif P. aeruginosa strain 8822 responds to any diffusable metabolite fromthe cancer cells. No secretion of azurin was observed under suchconditions, demonstrating that azurin secretion was contact-dependent,but energy independent for secretion. Such a mode of secretiondistinguishes azurin secretion on exposure to cancer cells from othermodes of protein secretion. Kostakioti et al., J. Bacteriol.187:187:4306-4314 (2005); Thanassi et al., Mol. Membr. Biol. 22:63-72(2005).

Example 3 MCF-7 Cells Specifically Induce the Release of Azurin from E.coli

Azurin is a periplasmic protein in P. aeruginosa. When the P. aeruginosaazurin gene is expressed in Escherichia coli JM109, the resultant azurinprotein is found in the periplasm and is purified from the periplasmicfractions of E. coli cells. Goto et al., Mol. Microbiol. 47:549-559(2003). Another periplasmic protein of P. aeruginosa is cytochrome c₅₅₁which acts as a partner of azurin during electron transfer in vitro andwhich also can be purified from the periplasmic fraction of E. coliJCB7120 cells grown under anaerobic conditions. Id.

The secretion of azurin and cytochrome c₅₅₁ from both P. aeruginosa andE. coli strains in absence or in presence of MCF-7 cells wasinvestigated. This investigation determined if exposure to MCF-7 breastcancer cells allows disruption of the bacterial outer membrane torelease any periplasmic protein or if there is specificity in thesecretion of azurin. Further, this investigation determined if azurinsecretion is specific for P. aeruginosa or could occur from otherbacteria such as E. coli.

The level of intracellular or extracellular cytochrome c₅₅₁ in P.aeruginosa 8822 under the defined conditions of aerobic growth was toolow to be detectable by Western blotting. However, both azurin andcytochrome c₅₅₁ were clearly detectable in the extracts of the E. colicells that hyper-expressed the proteins (FIG. 3, A and B, time 0*).Similar to P. aeruginosa, exposure of E. coli cells harboring the P.aeruginosa azu gene to MCF-7 cells elicited significant secretion of theazurin to the external medium, but only in presence of the cancer cells(FIG. 3A). Interestingly, however, when the E. coli cells harboringperiplasmic cytochrome c₅₅₁ were exposed to MCF-7 cells, very littlecytochrome c₅₅₁ was detected in the external growth medium (FIG. 3B),suggesting that exposure to MCF-7 cells specifically induces azurin, butnot cytochrome c₅₅₁, secretion from E. coli even when intracellularcytochrome c₅₅₁ was clearly detectable (FIG. 3B).

Example 4 Secretion of Azurin from P. aeruginosa or E. coli isEnergy-Independent

To determine if azurin secretion from either P. aeruginosa 8822 or E.coli JM109 required energy, the bacteria were incubated in presence ofthe protonophore carbonyl cyanide chlorophenylhdrazone (CCCP) for 1 hrat two different concentrations (50 and 250 μM for P. aeruginosa 8822;250 μM for E. coli JM109) before exposure to the MCF-7 cancer cells.CCCP is an uncoupler of oxidative phosphorylation and inhibitedbacterial growth at the concentrations used (FIG. 3E; left panel, P.aeruginosa 8822; right panel, E. coli JM109). Nevertheless, CCCP hadvery little effect on azurin secretion by either P. aeruginosa 8822(FIG. 3C) or E. coli JM109 (FIG. 3D), indicating the energy-independenceof azurin secretion.

Example 5 MCF-7 Cells Induced the Release of 15 kb DNA from P.aeruginosa

Bacterial DNA is known to be released or transferred during conjugationin response to cellular demands, using type IV secretion systems.Cascales and Christie, Nature Rev. Microbiol. 1:137-149 (2003). In sucha system, direct transfer of proteins and DNA may occur in target hostcells from the cytoplasm as well as from the periplasm of the donorbacteria. However, the presence of the host cells or the dependence onhost cell contact is not mandatory because protein or chromosomal DNAsecretion by type IV system into the growth medium in absence of anyhost cells has also been demonstrated. Burns, Curr. Opin. Microbiol.2:25-29, 1999; Hamilton et al., Mol. Microbiol. 55: 1704-1721 (2005). InP. aeruginosa, released DNA is known to contribute to theproinflammatory processes during infection in the lungs of cysticfibrosis patients or during formation of biofilms. Delgado et al.,Infect. Immun. 74: 2975-2984 (2006); Whitchurch et al., Science 295:1487(2002). The release of any specific CpG-rich extrachromosomal DNA,however, has not been reported.

In an effort to see if P. aeruginosa strain 8822 might release into itsgrowth medium not only azurin but also genomic DNA, the growth medium ofstrain 8822 was examined during growth for 1 or 2 hr under conditionswhere very little azurin was seen in absence of exposure to MCF-7 cells.To detect the presence of any nucleoprotein complex, 3 volumes ofnormal-isopropanol were added to the growth media followed by incubationat −80° C. for 1 hr. The nucleoprotein pellet was centrifuged,resuspended in double-distilled water and passed through the Qiagen® DNApreparation kit (Qiagen, Inc., Valencia, Calif.) before elution withdouble-distilled water. While there was no DNA detected at 0 h (start ofthe experiment), a specific band of DNA of about 15 kb size was detectedat 1 and 2 h, both in absence or in presence of MCF-7 cells (FIG. 4A).

The amount of DNA was higher in presence of the cancer cells, suggestingenhanced release as is the case with azurin, but in contrast to azurin,the DNA could be detected even in the absence of the cancer cellspresumably because of a much higher sensitivity for detection, usingPicoGreen® (Invitrogen, Inc., Carlsbad, Calif.) and PCR reactions.Interestingly, no other significant DNA bands were seen, and this bandis highly reproducible under repeated experimental conditions,suggesting that it is not a random product of chromosomal DNA digestion.

Example 6 Release Profile Suggests 15 kb DNA is Extrachromosomal

Genomic islands carrying virulence-associated genes are well-known in P.aeruginosa. Kiewitz et al., Microbiology 146: 2365-2373 (2000); He etal., Proc. Natl. Acad. Sci. 101:2530-2535 (2004); Klockgether et al., J.Bacteriol. 186:518-534 (2004); Kulasekara et al., J. Bacteriol.188:4037-4050 (2006). Biodegradative gene clusters conferring selectivegrowth advantage in xenobiotic-contaminated environments are also knownas mobile genetic elements in Pseudomonas species that are oftenassociated with phage genes. van der Meer et al., Arch. Microbiol.175:79-85 (2001). A single genomic island, PAGI-1, having at least twodifferent DNA segments of G+C content 63.7% and 54.9% with genesinvolved in possible detoxification of reactive oxygen species, has beenshown to be present in a number of clinical isolates of P. aeruginosa.Liang et al., J. Bacteriol. 185:843-853 (2001).

Similar to azurin secretion, the kinetics of the release of this DNAfragment showed its extracellular presence as early as in 5 min (FIG.4B), even in absence of MCF-7 cells. No other DNA bands were seen undersuch conditions. A kinetic study of the release of azurin and the 15 kbDNA fragment in absence of any other chromosomal DNA fragment onexposure to MCF-7 cells demonstrated a similar time course of release(FIG. 4C), suggesting that the DNA is an extrachromosomal element,perhaps looping out of the chromosome as a horizontally acquired genomicisland.

Example 7 15 kb DNA Release from P. aeruginosa is CpG-Rich DNA

In order to examine the nature of the released DNA, the DNA wassubjected to various restriction endonuclease digestions. Interestingly,only MSP-1 and PvuI, which are known to cleave between G and C residues,induced extensive digestion of the DNA, indicating that the DNA is richin G+C (FIG. 4D). When fragments of the partial digestions of theserestriction enzymes, or mechanically sheared fragments, were sequencedand the homologies of the sequences were compared with those in thedatabases, several DNA sequences both present and absent in the P.aeruginosa PAO genome, were seen (FIG. 7) and SEQ ID NOS: 26-62,suggesting that the released DNA came from a genomic island in strain8822. No plasmid DNA isolated by the usual methods of isolation has beenobtained. An interesting sequence present in the CpG-rich DNA is astretch of cytosines (FIG. 5A) and with many CpG dinucleotide sequencesin it.

Example 8 The 15 kb CpG-Rich DNA Band Contains a Sequence Similar toAzurin

An interesting sequence present is that of the azurin gene, butresembling the azurin gene from Neisseria and demonstrating 95%nucleotide sequence identity with it. The amino acid sequencecomparisons are shown in FIG. 5B. Since the Neisseria) azurin gene hasan additional DNA sequence in the 5′-end, encoding the 39 amino acid H.8epitope at the N-terminal of azurin, the CpG-rich released DNA was usedas a template and both the H.8 and the azurin gene sequences were usedas well as the whole gene from the Neisseria) H.8-azurin gene calledlaz. Hong et al., Cell Cycle 5:1633-1641 (2006). All three fragmentscould be amplified by PCR, demonstrating the presence of both thecomponents of the Neisseria) laz gene in the CpG rich DNA.

Example 9 The Released CpG-Rich DNA Activates TLR9-Mediated NF-kB andhas Antitumor Property

CpG deoxyoligonucleotides have been reported to have antitumorproperties. Krieg, Nature Med. 9:831-835 (2003); Krieg, Curr. Oncol.Rep. 6:88-95 (2004). To determine if the 15 kb CpG-rich DNA releasedfrom P. aeruginosa strain 8822, has properties similar to CpG syntheticoligodeoxynucleotides (ODNs), the ability of the DNA to activate NF-kBin a TLR9-dependent manner was tested. HEK293 cells, deficient in TLR9,were transfected with a TLR9-expressing plasmid. Kandimalla et al.,Proc. Natl. Acad. Sci. USA 102:6925-6930 (2005). A pNIFty plasmidexpressing the SEAP (secreted embryonic alkaline phosphatase) under thecontrol of an NF-kB inducible ELAM1 composite promoter (Invitrogen,Inc., Carlsbad, Calif.) was then used. SEAP expression following NF-kBactivation was measured in supernatants of transfected HEK293 cells,using the SEAP reporter assay kit (Invitrogen, Inc., Carlsbad, Calif.).The SEAP reporter assay was conducted as described by Schindler andBaichwal (Mol. Cell. Biol. 14:5820-5831 (1994)).

There was very little expression of the NF-kB-promoter inducible SEAP inthe TLR9-deficient HEK293 cells (FIG. 6A). However, inTLR9-expressing-HEK293 cells, there was increasing SEAP activity withincreasing amounts of the CpG-rich 15 kb DNA, suggesting that the DNAcould activate the NF-kB promoter only in TLR9-proficient cells (FIG.6A).

To determine if the activation of TLR9-dependent NF-kB results in tumorcell death, the expression of TLR9 was measured in a range of cancercells. Essentially all the cancer cell lines, the prostate cancer DU145,the breast cancer MCF-7 and the lung cancers H23 and A549 expressed TLR9and other Toll-like receptors like TLR4 (FIG. 6B). When the CpG-rich DNAwas incubated with the lung cancer cells A549 at increasingconcentrations for 12 and 24 h, there was increasing, albeit limited,cell death, suggesting that the CpG-rich DNA had low antitumor activity(FIG. 6C).

Example 10 Efficacy Study

A double-blind, placebo controlled study will be conducted over asix-month period. A total of 80 subjects with cancer, aged 45-60 years,will be chosen for the study. Primary recruitment of subjects will occurthrough physician referrals. Subjects who are successfully prescreenedwill be contacted by telephone and invited to a screening/orientationsession. Prospective subjects will be provided with a verbal and writtendescription of the study and requirements for participation. Writteninformed consent will be obtained from each participant at thescreening/orientation session, and medical record releases will beobtained and reviewed by the project oncologist. Participants givingconsent will be assigned a study ID number. During the screening sessionthe following information will be collected:

Demographics: Name, address, phone number, primary physician andoncologist, date of birth, race, ethnicity, occupation and years ofcompleted education.

Medical history: Current and past medical conditions including anassessment of cancer status, medications and supplements,hospitalizations, surgery, allergies, tobacco, alcohol and illicit druguse.

Basic Physical Examination: Height, weight, blood pressure, pulsereadings, and examination of the breasts, heart, lungs and abdomen.

Blood samples: Negative pregnancy test and normal liver function testing(ALT, AST) and hemoglobin.

The 80 subjects will be separated into 2 separate groups of 40 subjects.The first group will be administered one dosage form comprising SEQ IDNO: 26 and a pharmaceutically acceptable carrier once a day. The secondgroup will be administered one dosage form of placebo once a day. Bodyweight and tumor volume (including tumor progression, stasis, regressionand multiplicity) will be monitored every 3 days during the treatmentperiod. The following tumor volume categories will be used for scoring:Progression: the tumor grows more than 40% in area compared tocommencement of treatment; Stasis: the tumor did not fluctuate more than40% from its initial area throughout the course of treatment;Regression: the tumor regressed more than 40% from its initial area;Multiplicity: appearance of new tumors during the treatment.

Body Weight. There will be an observable difference in body weightbetween the subjects receiving SEQ ID NO: 26 and a pharmaceuticallyacceptable carrier versus placebo. Those receiving SEQ ID NO: 26 and apharmaceutically acceptable carrier will, on average, weigh more thanthose receiving placebo when height and sex is controlled for.

Tumor Volume. At the beginning of treatment, tumor sizes in both groupswill be comparable and not significantly different from each other. Onaverage, the tumor volume of placebo subjects will increase at avariable rate. Subjects receiving SEQ ID NO: 26 and a pharmaceuticallyacceptable carrier will show reduced growth in tumor size when comparedto placebo treated subjects.

Tumor Progression. The percent of tumors that progress will becalculated as described above. Subjects receiving SEQ ID NO: 26 and apharmaceutically acceptable carrier will demonstrate less tumorprogression over the course of the study than subjects receivingplacebo.

Tumor Stasis. The percent of tumors that remain in stasis will becalculated as described above. Subjects receiving SEQ ID NO: 26 and apharmaceutically acceptable carrier will demonstrate more tumor stasisas compared to tumor growth than subjects receiving placebo.

Tumor Regression. The percent of tumors that regress will be calculatedas described above. Subjects receiving SEQ ID NO: 26 and apharmaceutically acceptable carrier will demonstrate more tumorregression than subjects receiving placebo.

Tumor Multiplicity. The percent of tumors that multiply will bedetermined as described above. Subjects receiving SEQ ID NO: 26 and apharmaceutically acceptable carrier will demonstrate less tumormultiplicity than subjects receiving placebo.

Although the illustrated embodiments of the present invention have beendescribed herein with reference to the accompanying drawings, it isunderstood that the invention is not limited to those preciseembodiments and that various other changes and modifications may beeffected thereon by one skilled in the art without departing from thescope or spirit of the invention, and that it is intended to claim allsuch changes and modifications as fall within the scope of theinvention.

What is claimed is:
 1. A pharmaceutical composition comprising: apolynucleotide comprising a sequence selected from the group consistingof: SEQ ID NOS: 36, 38-41 and 43-45, wherein the polynucleotide has beenstabilized by condensation or spray-dried to produce a particulate formin one or a combination of shapes selected from the group consisting ofspherical, rod and toroid; and a pharmaceutically acceptable carrier;wherein the polynucleotide remains in the particulate form in thepharmaceutically acceptable carrier.
 2. The pharmaceutical compositionof claim 1 which additionally comprises at least one cupredoxin peptide.3. The pharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition is formulated for intravenous administration.
 4. Thepharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition is formulated for subcutaneous administration.
 5. Thepharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition is formulated for topical administration.
 6. Thepharmaceutical composition of claim 1, wherein the particulate form hasa size in the range of about 20 nm to about 500 nm.
 7. Thepharmaceutical composition of claim 1, wherein the particulate form ishollow.
 8. The pharmaceutical composition of claim 1, wherein theparticulate form is solid.